Ehu Camille Aka

Eric Wimmer

Elvina Barré

Daniel Cortés-Borda

Tchirioua Ekou

Lynda Ekou

Mireia Rodriguez-Zubiri

François-Xavier Felpin

Comparing Gas-Liquid Segmented and Tube-in-Tube Setups for the Aerobic Dimerization of Desmethoxycarpacine with an Automated Flow Platform

Keywords: Flow chemistry, Gas/liquid segmented oxidation, Tube-in-tube, Benzoxanthenone

The hazardous nature of molecular oxygen can be mitigated with the use of flow reactors of small size which, in turn, accelerate the gas to liquid mass transfer rate. In this contribution, we describe a reconfigurable automated flow platform integrating an online HPLC monitoring for the cobalt-catalyzed aerobic oxidative dimerization of desmethoxycarpacine 1 to carpanone 2. The flow platform was configured for aerobic oxidation under either a gas-liquid segmented or tube-in-tube strategy. An extensive optimization study concluded that the tubein-tube device was penalized by the use of expensive and fragile Teflon AF-2400 tubing while the gas-liquid segmented setup featured higher yields (62% vs. 48%), faster reaction rates (5 min vs. 20 minutes) and better robustness.

■ INTRODUCTION

Catalytic aerobic oxidations using molecular oxygen are attractive transformations which often meet the main principles of sustainable chemistry. [START_REF] Bäckvall | Modern Oxidation Methods[END_REF][START_REF] Punniyamurthy | Recent Advances in Transition Metal Catalyzed Oxidation of Organic Substrates with Molecular Oxygen[END_REF][START_REF] Parmeggiani | Transition metal based catalysts in the aerobic oxidation of alcohols[END_REF][START_REF] Shi | Recent advances in transition-metal catalyzed reactions using molecular oxygen as the oxidant[END_REF][START_REF] Campbell | Overcoming the "Oxidant Problem": Strategies to Use O2 as the Oxidant in Organometallic C-H Oxidation Reactions Catalyzed by Pd (and Cu)[END_REF] Benefits of using oxygen are twofold since it is a relatively inexpensive oxidant and can be readily removed from reaction mixtures. Simplified work-up procedures and cleaner syntheses are the main consequences of these features. However, due to its inherent hazardous nature associated to the potential risks of fire and explosion, the handling of oxygen requires the use of stainless-steal autoclaves with a high resistance to pressure in a well ventilated dedicated area. [START_REF] Osterberg | Experimental Limiting Oxygen Concentrations for Nine Organic Solvents at Temperatures and Pressures Relevant to Aerobic Oxidations in the Pharmaceutical Industry[END_REF] Another critical point associated with the use of reactive gas, including oxygen, is the low gas to liquid mass transfer rate which strongly affects kinetics, especially upon scaling-up. [START_REF] Kurt | Enhanced gas-liquid reactions[END_REF] Therefore, chemists are often reluctant to develop experimental sequences using oxygen and prefer the use of nongaseous oxygen surrogates which include atom inefficient, toxic and environmentally deleterious stoichiometric metal oxides, hypervalent iodine compounds and hydroperoxides.

Most of the issues associated with the use of oxygen can be addressed by switching from traditional batch synthesis to continuous flow chemistry. [START_REF] Hone | The Use of Molecular Oxygen for Liquid Phase Aerobic Oxidations in Continuous Flow[END_REF][START_REF] Mallia | The Use of Gases in Flow Synthesis[END_REF][START_REF] Gavriilidis | Aerobic oxidations in flow: opportunities for the fine chemicals and pharmaceuticals industries[END_REF][START_REF] Gemoets | Liquid phase oxidation chemistry in continuous-flow microreactors[END_REF] The small size of channels in flow reactors mitigates the hazardous nature of oxygen, even under demanding conditions which are not accessible within batch reactors. [START_REF] Gutmann | Continuous-Flow Technology-A Tool for the Safe Manufacturing of Active Pharmaceutical Ingredients[END_REF][START_REF] Hessel | Novel Process Windows for Enabling, Accelerating, and Uplifting Flow Chemistry[END_REF] Also, the delivery of oxygen can be safely regulated and monitored with the use of a mass flow controller.

Aerobic oxidations in flow have been essentially achieved using two different approaches.

The most conventional strategy uses a segmented flow (Taylor flow regime), where liquid slugs are separated by segments of gas. [START_REF] Leclerc | Gasliquid selective oxidations with oxygen under explosive conditions in a micro-structured reactor[END_REF][START_REF] Pieber | Direct aerobic oxidation of 2-benzylpyridines in a gas-liquid continuous-flow regime using propylene carbonate as a solvent[END_REF][START_REF] Vanoye | A Safe and Efficient Flow Oxidation of Aldehydes with O2[END_REF][START_REF] Vanoye | Gas-Liquid Segmented Flow Microfluidics for Screening Copper/TEMPO-Catalyzed Aerobic Oxidation of Primary Alcohols[END_REF][START_REF] Vanoye | Continuous, Fast, and Safe Aerobic Oxidation of 2-Ethylhexanal: Pushing the Limits of the Simple Tube Reactor for a Gas/Liquid Reaction[END_REF][START_REF] Cherkasov | Counting bubbles: precision process control of gas-liquid reactions in flow with an optical inline sensor[END_REF][START_REF] Aand | Controlled photo-flow oxidative reaction (UV-FOR) platform for ultra-fast phthalide and API synthesis[END_REF][START_REF] Feng | Visualization of two-phase reacting flow behavior in a gas-liquid-solid microreactor[END_REF][START_REF] Kumar | Integrated Microfluidic Photo-Flow Process (μ-PFP) for Direct Upconversion of Exhaust Gas to Value-Added Chemicals[END_REF] The liquid slugs are subjected to toroidal vertices which increase the interfacial area and improve the gas-liquid mass transfer. [START_REF] Yang | Simulations and analysis of multiphase transport and reaction in segmented flow microreactors[END_REF] Another approach, allowing efficient gas-liquid contact, consists in the use of a gas-permeable tubing (Teflon AF-2400) inserted inside a larger diameter tubing. [START_REF] O'brien | Flow Ozonolysis Using a Semipermeable Teflon AF-2400 Membrane To Effect Gas-Liquid Contact[END_REF][START_REF] Brzozowski | Flow Chemistry: Intelligent Processing of Gas-Liquid Transformations Using a Tube-in-Tube Reactor[END_REF][START_REF] Petersen | The Oxygen-Mediated Synthesis of 1,3-Butadiynes in Continuous Flow: Using Teflon AF-2400 to Effect Gas/Liquid Contact[END_REF][START_REF] Bourne | A Continuous Flow Solution to Achieving Efficient Aerobic Anti-Markovnikov Wacker Oxidation[END_REF] The gas can diffuse across the permeable membrane through the formation of microbubbles on the wall of the Teflon AF-2400 tubing which are dissolved into the flowing liquid. [START_REF] Yang | Mass Transport and Reactions in the Tube-in-Tube Reactor[END_REF] We have been interested by the bio-inspired cobalt-catalyzed oxidative dimerization of 2propenyl phenols in flow [START_REF] Cortés-Borda | An Autonomous Self-Optimizing Flow Reactor for the Synthesis of Natural Product Carpanone[END_REF][START_REF] Aka | Reconfigurable Flow Platform for Automated Reagent Screening and Autonomous Optimization for Bioinspired Lignans Synthesis[END_REF] which led to the formation of natural lignans sharing a benzoxanthenone structure (Figure 1). [START_REF] Chapman | Total Synthesis of Carpanone[END_REF][START_REF] Baxendale | A Concise Synthesis of Carpanone Using Solid-Supported Reagents and Scavengers[END_REF][START_REF] Daniels | A New Catalytic Cu(II)/Sparteine Oxidant System for β,β-Phenolic Couplings of Styrenyl Phenols: Synthesis of Carpanone and Unnatural Analogs[END_REF][START_REF] Liron | A New Cross-Coupling-Based Synthesis of Carpanone[END_REF][START_REF] Constantin | Oxidative Dimerization of (E)-and (Z)-2-Propenylsesamol with O2 in the Presence and Absence of Laccases and Other Catalysts: Selective Formation of Carpanones and Benzopyrans under Different Reaction Conditions[END_REF][START_REF] Matsumoto | Transition Metal(II) Schiff's Base Complexes Catalyzed Oxidation of trans-2-(1-propenyl)-4,5-methylenedioxyphenol to Carpanone by Molecular Oxygen[END_REF] In our initial studies, we carried out the oxidative dimerization using a stock solution of cobalt catalyst in 1,2-dichloroethane saturated with oxygen. [START_REF] Cortés-Borda | An Autonomous Self-Optimizing Flow Reactor for the Synthesis of Natural Product Carpanone[END_REF] While the experimental simplicity was the obvious asset of this procedure, we faced critical issues upon scaling-up. First, the experimental setup required to pre-saturate the stock solution with O 2 but without any possibility to further introduce O 2 in the reactor coil. Such approach required to work at a low concentration of starting material to prevent oxygen to be the limiting reagent, hampering the reaction productivity. The second issue was related to safety concerns associated with the handling of several liters of highly flammable stock solutions. We latter investigated non-gaseous oxygen surrogates using an automated screening platform and we identified t-BuOOH as a very efficient oxidant which significantly improved kinetics. [START_REF] Aka | Reconfigurable Flow Platform for Automated Reagent Screening and Autonomous Optimization for Bioinspired Lignans Synthesis[END_REF] However, the relatively high cost of t-BuOOH with respect to oxygen, prompted us to deeply investigate both the segmented flow and tube-in-tube technologies for the oxidative dimerization of desmethoxycarpacine 6 to carpanone 1.

■ RESULTS AND DISCUSSION

An automated flow platform including the tube-in-tube technology was set up to screen a variety of experimental conditions on the µmol scale (Figure 2). A computer, running an ad-hoc process control software, controls the automated modules through RS-232 ports (represented by red dashed lines). Our initial studies were first conducted with Co II (salen) complex 8a which was previously identified as an efficient catalyst for related transformations. [START_REF] Cortés-Borda | An Autonomous Self-Optimizing Flow Reactor for the Synthesis of Natural Product Carpanone[END_REF][START_REF] Constantin | Oxidative Dimerization of (E)-and (Z)-2-Propenylsesamol with O2 in the Presence and Absence of Laccases and Other Catalysts: Selective Formation of Carpanones and Benzopyrans under Different Reaction Conditions[END_REF][START_REF] Matsumoto | Transition Metal(II) Schiff's Base Complexes Catalyzed Oxidation of trans-2-(1-propenyl)-4,5-methylenedioxyphenol to Carpanone by Molecular Oxygen[END_REF] The results of our screening are depicted in Table 1. A rapid screening of the catalyst loading showed that working with 0.1, 1 or 5 mol% of complex 8a at a residence time of 10 minutes and with 2 bar of O 2 did not significantly affect the reaction outcome (Entries 4, 6, 11) while a sharp decrease of the reaction yield was observed when the catalyst loading was lowered to 0.05 mol% (Entry 2). An uncatalyzed pathway was also observed since the background reaction conducted in the absence of complex 8a furnished carpanone 1 with 13% yield (Entry 1). Surprisingly, when the residence time was increased from 10 to 20 or even 30 minutes, the reaction yield marginally increased, reaching a maximum of 48% at 1 mol% catalyst loading and 20 minutes of residence time (Entry 9). While this result cannot be fully rationalized, we hypothesized that it could be related to the rapid deactivation of the Co II (salen) complex 8a in the presence of oxygen into inactive and insoluble Co species. At high catalyst loadings (≥ 1 mol%) the formation of insoluble particles was fast and were visually detected through the transparent tubing. The presence of insoluble Co species were attributed to the formation cobalt clusters which were favored at higher concentration. A related phenomenon was reported in palladium catalysis. [START_REF] Reetz | Ligand-free Heck reactions using low Pd-loading[END_REF] A modulation of the pressure of oxygen provided interesting insights. A decrease of the pressure from 2 to 1 bar resulted in a sharp drop of the reaction yield from 43% to 9% (Entry 7) while, on the contrary, an increase of the pressure to 4 bar did not affect the reaction outcome (Entry 8). This result suggested that the mass transfer of O 2 to the liquid stream at pressure >2 bar is not a limiting factor. Three other Co II complexes (8b-d) were also evaluated, but without significant success (Entries 13-15). Interestingly, switching oxygen for air did not erode the reaction yield which reached a maximum of 47% at 20 min of residence time versus 48% with oxygen (Entries 16-17), corresponding to a space time yield of ca. 3.6 g.h -1 .L -1 . This result confirms that the oxidative dimerization of desmethoxycarpacine 6 to carpanone 1 with the tube-in-tube technology is neither governed by the mass transfer of gas to liquid nor by the formation of a radial gradient of gas with the highest concentration near the wall of the Teflon AF-2400 tubing. The modest reaction yields obtained with the tube-intube strategy was attributed to the tendency of Co complex 8a to aggregate into inactive species. We suspected that this behavior is accelerated by the absence of efficient mixing which was mainly governed by diffusion phenomenon. One of the main limitations of the conventional tube-in-tube technology is the impossibility to conduct heated reactions because the flowing liquid is protected from the heat by a shell of gas. To circumvent this drawback, a reverse tube-in-tube configuration in which the liquid flows on the shell side and the gas is introduced through the inner Teflon AF-2400 tubing, has been occasionally reported. [START_REF] Mercadante | Continuous-flow, palladium-catalysed alkoxycarbonylation reactions using a prototype reactor in which it is possible to load gas and heat simultaneously[END_REF][START_REF] Mercadante | Continuous Flow Hydrogenation Using an On-Demand Gas Delivery Reactor[END_REF] We envisioned the oxidative dimerization of desmethoxycarpacine 6 to caparnone 1 using the reverse tube-in-tube design. Unfortunately, in our preliminary experiments we faced an extensive diffusion phenomenon within the liquid stream which significantly increased the residence time compared to the normal tube-in-tube design at a given flow rate and strongly modified the reaction concentration as evidenced by the in-line UV monitoring. This unexpected behavior prompted us to give up on this strategy.

The automated tube-in-tube platform was reconfigurated for conducting gas-liquid segmented flow reactions (Figure 4). The tube-in-tube device was replaced by a single 1/8" ETFE tube (7.8 mL, 1.72 m, 3.2 mm o.d., 2.4 mm i.d.) connected at the inlet to the liquid stream containing a mixture of desmethoxycarpacine 6 (0.1 M) and p-xylene 7 (1 M) in DCE and the gas inflow through a PEEK T-shaped junction with a stoichiometric molar ratio of ca.

3 (O 2 / desmethoxycarpacine 6). The gas stream was precisely delivered through the use of a mass flow controller. The reactor outlet was connected to an automatic 2-way-6-port switch valve which injected an aliquot of the reaction mixture into the HPLC line. The gas-liquid stream was maintained at a constant pressure of 4 bar with an electronic back pressure regulator. The use of a mass flow controller associated to the low volume of gas in the ETFE reactor, estimated to be below 6 mL at any given time, minimized the risks associated with the use of O 2 . The liquid handling robot which prepared the reaction mixtures, the heating chamber and the HPLC analysis system were connected to the computer through RS-232 ports (red dashed lines) and controlled with MATLAB. Based on the previous results collected with the tube-in-tube system, we only considered the use of cobalt complex 8a to evaluate the gas-liquid segmented approach. Using O 2 as oxidant, we evaluated the influence of the loading of complex 8a on the reaction yield for a given residence time of 10 minutes (Table 2). The reaction yield reached a plateau at ca. 62-65% with a loading of 8a 1 mol% (Entries 2-6), while the background reaction conducted in the absence of catalyst 8a marginally produced carpanone 1 (6% yield, Entry 1).

One advantage of the gas-liquid segmented system compared to the tube-in-tube device is the possibility to heat the reaction mixture. However, in our hand, the temperature increase from 25 to 45 °C was detrimental to the oxidative dimerization of desmethoxycarpacine 6 to carpanone 1. The high instability of both desmethoxycarpacine 6 and catalyst 8a under heating are likely responsible of this behavior. Similarly, an increase of the residence time from 10 to 20 minutes did not affect the reaction yield (Entry 9) while a decrease of the residence time to 5 minutes was not detrimental and furnished carpanone 1 with 62% yield, corresponding to a space time yield of ca. 4.7 g.h -1 .L -1 . Switching oxygen for air did not significantly modified the reaction outcome, though a higher catalyst loading (2.5 mol% 8a) and increased residence (10 minutes) were required to reach the highest reaction yield (62%, Entry 12). As already observed with the use of oxygen, any changes on experimental conditions failed to improve the reaction outcome since an increase of the temperature to 45 °C altered the yield (48%, Entry 13) while an increase of the residence time to 20 minutes had no impact (Entry 15). These last results suggest that the catalytic cycle stops after ca. 5-10 minutes due the high sensitivity of complex 8a which rapidly degrades under our experimental conditions. While our automated screening platform is not suited for scale-up experiments, it can be to prepare up to gram quantities through either higher volumes of injection (up to 2 mL per injection) or successive injections. For instance, we repeated 10 times the injection of 2 mL of the crude mixture from entry to isolate after purification 464 mg of carpanone 1 with an average yield of 61% (average over ten reactions). From these studies we learnt a number of interesting features. Whatever the strategy used, tube-in-tube versus segmented flow, the gas to liquid mass transfer was not identified as the rate limiting factor. The high interfacial area per volume offered in flow reactors explained that the gas-liquid mass transfer was not the rate limiting step in our study. The switch of oxygen to air did not significantly affect the reaction outcome. This interesting feature could greatly facilitate the scaling-up as the use of air significantly mitigates the risks associated with such process. The gas-liquid segmented approach proved to be superior to the tube-intube system in terms of both yield (48 vs. 62%) and residence time (20 vs. 5 minutes) for the oxidative dimerization of desmethoxycarpacine 6 to carpanone 1. While both approaches are of equivalent practicability, the gas-liquid segmented system was, in our hand, more robust than the tube-in-tube system toward the repetitive screening of experimental conditions. We were compelled to change the Teflon AF-2400 membrane ca. every two months of daily uses at pressure 4 bar because upon prolonged exposure to DCE it became fragile and ripped.

Moreover, Teflon AF-2400 tubing is a fragile perfluoropolymer which breaks upon folding, making tedious its handling. Another asset of the gas-liquid segmented approach is the selfmixing of liquid slugs due to the presence of vortices, also known as toroidal currents, which appear upon flowing despite a low Reynolds number ca. 5. While the rate of oxidations with gas are often affected by the gas to liquid mass transfer, we identified with both systems the catalyst deactivation the rate limiting step. The fast deactivation of complex 8a in the presence of oxygen and air bolsters our recent results showing the exceptional reactivity of t-BuOOH as oxygen-free surrogates for the cobalt-catalyzed dimerization of 3-propenylphenols into benzoxanthenones. 30

■ CONCLUSION

A reconfigurable automated flow platform has been designed for the cobalt-catalyzed oxidative dimerization of desmethoxycarpacine 6 to carpanone 1 with oxygen. The flow platform was used for the evaluation of two different approaches for the handling of molecular oxygen in flow. The traditional gas-liquid segmented strategy was compared to the original tube-in-tube system. We demonstrated that for both approaches, the gas to liquid mass transfer was not the rate limiting step of the cobalt-catalyzed oxidative dimerization and the catalyst deactivation was identified as a critical feature affecting the reaction yield. A careful assessment of both systems revealed that the gas-liquid segmented setup was more robust and allowed higher reaction yields than the tube-in-tube device which is penalized by the cost and fragility of the Teflon AF-2400 tubing. While we do not claim that the results compiled in this work can be generalized to any other oxidative process, they would serve to process chemists interested in the handling of oxygen in flow, far beyond the scope of the current study.

■ EXPERIMENTAL SECTION

General information. All commercially available chemicals were used as received unless otherwise noted.

■ ASSOCIATED CONTENT

The supporting information is available free of charge on the ACS Publication website at DOI: 10.1021/acs.oprd.xxxxxxx HPLC chromatograms; 1 H and [START_REF] Hessel | Novel Process Windows for Enabling, Accelerating, and Uplifting Flow Chemistry[END_REF] C NMR spectra of purified carpanone 2; photograph of the automated tube-in-tube system; photograph of the automated gas-liquid system; photograph of the gas-liquid segmented reactor (PDF)

■ AUTHOR INFORMATION

Corresponding author *E-mail: fx.felpin@univ-nantes.fr

ORCID

François-Xavier Felpin: 0000-0002-8851-246X

Notes

The authors declare no competing financial interest.

Figure 1 .

 1 Figure 1. Biomimetic synthesis of naturally-occurring benzoxanthenones.

Figure 2 .

 2 Figure 2. Automated tube-in-tube platform for the oxidative dimerization of desmethoxycarpacine 6 to carpanone 1. Structures of cobalt-catalysts evaluated with the tubein-tube approach.

Figure 3 .

 3 Figure 3. Photograph of the tube-in-tube device.

Figure 4 .

 4 Figure 4. Automated gas-liquid segmented flow platform for the oxidative dimerization of desmethoxycarpacine 6 to carpanone 1.

  [START_REF] Bäckvall | Modern Oxidation Methods[END_REF] H and[START_REF] Hessel | Novel Process Windows for Enabling, Accelerating, and Uplifting Flow Chemistry[END_REF] C NMR spectra were recorded at 300 or 400 and 75 MHz or 100 MHz, respectively.[START_REF] Bäckvall | Modern Oxidation Methods[END_REF] H and[START_REF] Hessel | Novel Process Windows for Enabling, Accelerating, and Uplifting Flow Chemistry[END_REF] C NMR spectra were referenced to the internal deuterated solvent (CDCl 3 ) at 7.26 and 77.16 ppm, respectively. FT-IR spectra were recorded in the ATR mode. Wavelengths of maximum absorbance (ν max ) are quoted in wave numbers (cm -1 ). High resolution mass spectrometry (HRMS) was recorded on a microTOF spectrometer equipped with orthogonal electrospray interface (ESI). Analytical thin-layer chromatography (TLC) was carried out on silica gel 60 F 254 plates and visualized with a UV lamp at 254 nm or stained with a basic potassium permanganate solution. Flash column chromatography was performed using silica gel 60 (40-63 µm). The AF-2400 permeable tubbing was purchased from Cambridge Reactor Design.Details of the experimental setup. HPLC pumps (JASCO PU2080) equipped with a RS-232 port were employed to flow the solution through the system. A sampler handler (JASCO AS 2055) equipped with a RS-232 port was used to inject the reagents in the line. The reactor coil was heated with a heating plate (Heidolph, MR Hei-Connect) equipped with a RS-232 port. The gas flow rate was controlled with a Brooks GF040 mass flow controller. A 2-way 6port valve (VICI, Cheminert C2-3006D) equipped with a RS-232 port was used to inject an aliquot of the crude mixture within the on-line HPLC unit. The HPLC column outlet was connected to a UV detector (JASCO, UV 2075) equipped with a RS-232 port. The flow outlet was connected to a programmable fraction collector (Advantec, CHF 1225C). All units equipped with a RS-232 port were autonomously controlled with MATLAB through the use of communication protocols provided by the manufacturers.Synthesis of Carpanone 2 through the tube-in-tube approach. An automatic sample handler prepared 200 µL of the reaction mixture from stock solutions of (E)-6-(prop-1-enyl)-1,3benzodioxol-5-ol 6[START_REF] Cortés-Borda | An Autonomous Self-Optimizing Flow Reactor for the Synthesis of Natural Product Carpanone[END_REF] (0.1 M + p-xylene 7 at 1 M) and [Co]-8a-d (7.5 × 10 -4 M). The reaction mixture was injected in a stream of C 2 H 4 Cl 2 pumped at 0.096 and 0.048 mL/min for 10 and 20 minutes residence time, respectively, as depicted in Figure1. The liquid stream entered into the inner Teflon AF-2400 tubing (0.96 mL, 1.9 m, 1 mm o.d., 0.8 mm i.d.) of the tube-in-tube device. The liquid stream in the inner tube was subjected to a pressure of 5 bar using an electronic backpressure regulator while the outer ETFE tube (7.1 mL, 1.9 m, 3.2 mm o.d., 2.4 mm i.d.) was subjected to 1, 2 or 4 bar of gas (oxygen or air) using a 2-stage regulator on the cylinder. The reactor outlet was connected to an automatic 2-way 6-port switch valve which injected 0.2 µL of the crude mixture in the HPLC unit while the remaining stream was collected in a fraction collector. A mixture of MeOH/H 2 O (70/30, v/v) was used as mobile phase for the HPLC analysis at a flowrate of 0.4 mL/min. A UV detector was connected to the outlet of the HPLC column (Agela Promosil C18, 3.5 mm × 150 mm, 5 µm) to follow the absorbance at a wavelength of 270 nm. Peak integration and yield calculation were under full MATLAB automation.Synthesis of Carpanone 2 through the gas-liquid segmented approach. An automatic sample handler prepared 200 µL of the reaction mixture from stock solutions of (E)-6-(prop-1-enyl)-1,3-benzodioxol-5-ol 6[START_REF] Cortés-Borda | An Autonomous Self-Optimizing Flow Reactor for the Synthesis of Natural Product Carpanone[END_REF] (0.1 M + p-xylene 7 at 1 M) and [Co]-8a (7.5 × 10 -4 M). The reaction mixture was injected in a stream of C 2 H 4 Cl 2 pumped at 0.38, 0.19 and 0.095 mL/min for 5, 10 and 20 minutes residence time, respectively, as depicted in Figure 3. The liquid stream was connected through a PEEK T-shaped piece (internal volume: 0.57 µL) to a second stream flowing oxygen or air at a flow rate of 1.18, 0.59 and 0.295 mL/min for 5, 10 and 20 minutes residence time, respectively. The flow rate of the gas stream is controlled by a mass flow controller. The gas-liquid segmented stream entered in a 1/8" ETFE reactor coil (7.8 mL, 1.72 m, 3.2 mm o.d., 2.4 mm i.d.) thermostated at the required temperature. The reactor outlet was connected to an automatic 2-way 6-port switch valve which injected 0.2 µL of the crude mixture in the HPLC unit while the remaining stream was collected in a fraction collector. The gas-liquid segmented flow was subjected to a pressure of 4 bar using an electronic backpressure regulator. A mixture of MeOH/H 2 O (70/30, v/v) was used as mobile phase for the HPLC analysis at a flowrate of 0.4 mL/min. A UV detector was connected to the outlet of the HPLC column (Agela Promosil C18, 3.5 mm × 150 mm, 5 µm) to follow the absorbance at a wavelength of 270 nm. Peak integration and yield calculation were under full MATLAB automation. Analytical data for carpanone 1. mp 191-192 °C [Lit.

Table 1 .

 1 Oxidative dimerization of desmethoxycarpacine 6 to carpanone 1 using the tube-intube technology described in Figure1. Unless otherwise stated, the pressure of gas was fixed to 2 bar. b The pressure of O 2 was fixed to 1 bar. c The pressure of O 2 was fixed to 4 bar.

	Entry a	Oxidant	Catalyst	[Co] loading	Residence	Yield (%)
				(mol %)	time (min)
	1	O 2	-	0	10	13
	2	O 2	[Co]-8a	0.05	10	27
	3	O 2	[Co]-8a	0.05	20	31
	4	O 2	[Co]-8a	0.1	10	40
	5	O 2	[Co]-8a	0.1	20	42
	6 7 b 8 c	O 2 O 2 O 2	[Co]-8a [Co]-8a [Co]-8a	1 1 1	10 10 10	42 9 43

Table 2 .

 2 Oxidative dimerization of desmethoxycarpacine 6 to carpanone 1 with catalyst [Co]-

	8a, using gas-liquid segmented approach.
	Entry	Oxidant	[Co] loading	Temperature	Residence	Yield (%)
			(mol %)	(°C)	time (min)
	1	O 2	0	25	10	6
	2	O 2	0.1	25	10	42
	3	O 2	0.5	25	10	55
	4	O 2	1	25	10	62
	5	O 2	2.5	25	10	62
	6	O 2	5	25	10	65
	7	O 2	2.5	45	10	54
	8	O 2	1	25	5	62
	9	O 2	1	25	20	60
	10	Air	0	25	10	5
	11	Air	1	25	10	40
	12	Air	2.5	25	10	62
	13	Air	2.5	45	10	48
	14	Air	2.5	25	5	58
	15	Air	2.5	25	20	61

  [START_REF] Constantin | Oxidative Dimerization of (E)-and (Z)-2-Propenylsesamol with O2 in the Presence and Absence of Laccases and Other Catalysts: Selective Formation of Carpanones and Benzopyrans under Different Reaction Conditions[END_REF] 189-190 °C]. IR (ATR) ν 2872, 1674, 1622, 1478, 1158, 1032, 910, 840 cm -1 . Hz), 2.52 (br qd, 1H, J = 7.2 Hz), 2.17-2.26 (m, 1H), 1.14 (d, 3H, J = 7.2 Hz), 0.71 (d, 3H, J = 7.6 Hz). 13 C NMR (75 MHz, CDCl 3 ) δ 187.0, 168.5, 146.8, 145.3, 143.3, 142.7, 126.4, 115.4, 107.2, 101.4, 100.5, 100.3, 99.4, 98.9, 36.4, 35.6, 35.4, 33.7, 21.6, 21.3. HRMS (ESI) m/z [M + H] + calcd for C 20 H 19 O 6 , 355.1182, found 355.1180.

1 H NMR (300 MHz, CDCl 3 ) δ 7.01-7.03 (m, 1H), 6.80 (s, 1H), 6.33 (s, 1H), 5.91 (d, 1H, J = 1.3 Hz), 5.88 (d, 1H, J = 1.3 Hz), 5.69 (s, 1H), 5.67 (s, 1H), 5.64 (s, 1H), 3.27 (dd, 1H, J = 2.3, 7.4 Hz), 3.19 (dt, 1H, J = 2.3, 7.4

■ ACKNOWLEDGEMENTS

We gratefully acknowledge the University of Nantes, the ''Centre National de la Recherche Scientifique'' (CNRS) and the "Région des Pays de la Loire" in the framework of the SmartCat project for financial support. E. C. Aka thanks the ''Ministère de l'Enseignement Supérieur et de la Recherche Scientifique de Côte d'Ivoire" for a visiting grant in France. We acknowledge Julie Hémez (CEISAM, University of Nantes) for HRMS analyses.


Hélène Dubrasquet

Ignacio Garrido

Paulina Bruning

Janette Reyes

Marie-Laure Guillemin
email: marielaure.guillemin@gmail.com

Building-up knowledge on green macroalgae diversity in the Western Antarctic Peninsula: data from two molecular markers reveals numerous species with amphipolar distribution

Keywords:

Low levels of diversity and endemism, when compared to red or brown algae, have been reported for Antarctic green macroalgae (Chlorophyta). However, recent studies including the use of molecular markers have allowed to revisit the taxonomical status of species thought to be well known, underlying the existence of unexpected Antarctic flora diversity at local and regional scale. In the present study, samples of green macroalgae along the Western Antarctic Peninsula (from the 62°S down to the 66°S) were sequenced for two genetic markers regularly used for species determination and barcoding in Chlorophyta (i.e. the plastid genes tufA and rbcL). From the 122 specimens of Chlorophyta sampled, 86 were sequenced for the gene tufA and 15 for the gene rbcL. Using the NCBI Nucleotide Blast Tool and a threshold of 95% of similarity to compare our sequences to the ones available in public data depositories allowed the identification of 12 species. Three new species were reported for the area:

Rosenvingiella radicans, Urospora wormskioldii and Ulvella islandica. Furthermore, molecular identification revealed strong match (> 95%) between our Antarctic sequences and the ones obtained for samples from the northern hemisphere for Acrosiphonia arcta, Prasiola crispa, Prasiola antarctica, R. radicans, Ulva sp. A-GW, U. islandica, Urospora penicilliformis and U. wormskioldii confirming the amphipolar distribution of various taxa of Antarctic Trebouxiophyceae and Ulvophyceae. Amphipolar distribution seems more common in green than red or brown Antarctic seaweeds, so here we hypothesize that recurrent occurrence of long dispersal events could explain the low level of endemism observed for this phylum along the Antarctic coasts.

INTRODUCTION

In green algae, the so-called "core" Chlorophyta (including the classes Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Pedinophyceae and Chlorodendrophyceae, [START_REF] Fang | -Improving phylogenetic inference of core Chlorophyta using chloroplast sequences with strong phylogenetic signals and heterogeneous models[END_REF], groups taxa that have diverged early from the Prasinophyceae during the Paleozoic era [START_REF] Fučíková | -New phylogenetic hypotheses for the core Chlorophyta based on chloroplast sequence data[END_REF][START_REF] Leliaert | -Phylogeny and Molecular Evolution of the Green Algae[END_REF] and actually comprise 6,500 species (Guiry & Guiry, 2019). These species are ecologically and morphologically very diverse and are found in a wide variety of marine and freshwater environments. In the cold waters surrounding Antarctica, fifteen to seventeen species belonging to Ulvophyceae, Trebouxiophyceae and Chlorophyceae have historically been reported [START_REF] Gallardo | -New collections of benthic marine algae from Livingston and Deception Islands (South Shetland Islands) and Trinity Island (Bransfield Strait) Antarctica[END_REF][START_REF] Ramírez | -Flora marina bentónica de la región austral de Sudamérica y la Antártica[END_REF][START_REF] Wiencke | Antarctic Seaweeds[END_REF][START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF]. However, the recent study of [START_REF] Pellizzari | Diversity and spatial distribution of seaweeds in the South Shetland Islands, Antarctica: an updated database for environmental monitoring under climate change scenarios[END_REF] updated this number to 24 along the coasts of the South Shetland Islands (SShs), with five new records for the area (Chaetomorpha (Lola) irregularis, Rhizoclonium ambiguum, Monostroma grevillei, Spongomorpha (Acrosiphonia) arcta and Ulvella viridis), and two putative new species (Callophyllis sp. and Prasiola sp. distinct from Prasiola crispa already mentioned for the area). Some of these new records are supported by results obtained with molecular markers (i.e. cytochrome c oxidase -COI-5P, UPA genes and Internal Transcribed Spacer -ITS -region for Callophyllis sp., M. grevillei Protomonostroma sp. and Prasiola sp.).

Recent studies including molecular tools have allowed to revisit the taxonomical status of species thought to be well known, improving knowledge on diversity and level of endemism characterizing the Antarctic flora (red algae: [START_REF] Dubrasquet | -Molecular-Assisted Revision of Red Macroalgal Diversity and Distribution along the Western Antarctic Peninsula and South Shetland Islands[END_REF]Guillemin et al., 2018;[START_REF] Hommersand | -Notes on the systematics and biogeographical relationships of Antarctic and sub-Antarctic Rhodophyta with descriptions of four new genera and five new species[END_REF][START_REF] Ocaranza-Barrera | -Molecular divergence between Iridaea cordata (Turner) Bory de Saint-Vincent from the Antarctic Peninsula and the Magellan Region[END_REF]Pellizzarri et al., 2017;green algae: De Wever et al., 2009;[START_REF] Garrido-Benavent | -From Alaska to Antarctica: Species boundaries and genetic diversity of Prasiola (Trebouxiophyceae), a foliose chlorophyte associated with the bipolar lichen-forming fungus Mastodia tessellata[END_REF]Moniz et al., 2012;Pellizzarri et al., 2017;brown algae: Peters et al., 1997;[START_REF] Peters | -The phylogenetic position of the subantarctic marine macroalga Desmarestia chordalis (Phaeophyceae) inferred from nuclear ribosomal ITS sequences[END_REF]. In Chlorophyta, studies using plastid sequences have underlined unexpected diversity at local and regional scale along the Antarctic coasts for Chlorophyceae and Trebouxiophyceae (DeWever et al., 2009;[START_REF] Moniz | [END_REF] and pointed out that the supposedly well-known Antarctic green macroalgae diversity, with very few species reported in comparison with other marine realms [START_REF] Griffiths | -Antarctic Marine Biodiversity -What do we know about the distribution of life in the Southern Ocean?[END_REF], could be underestimated (DeWever et al., 2009;Moniz et al., 2012;[START_REF] Mystikou | Seaweed biodiversity in the southwestern Antarctic Peninsula: surveying macroalgal community composition in the Adelaide Island/Marguerite Bay region over a 35-year time span[END_REF]. However, few molecular data are available in public data repository for Antarctic green algae (i.e. 70 sequences of macroalgae obtained as result for a search for "Antarctic marine Chlorophyta" in Genbank database considering all available molecular markers, author's person. obs.).

Accurate and exhaustive understanding of the native algal flora biodiversity and distribution is a key factor for monitoring Antarctic seaweeds [START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF]. In the Western Antarctic Peninsula (WAP), recent transformations of the physical environment linked to global climate change (e.g. increasing sea temperatures and sea ice melting: [START_REF] Etourneau | -Ocean temperature impact on ice shelf extent in the eastern Antarctic Peninsula[END_REF][START_REF] Holland | -West Antarctic ice loss influenced by internal climate variability and anthropogenic forcing[END_REF]Meelh et al., 2019;[START_REF] Valdivia | Biodiversity of an Antarctic rocky subtidal community and its relationship with glacier meltdown processes[END_REF] may for example favor the arrival and settlement of non-native species, affecting the whole benthic communities functions [START_REF] Hughes | Invasive non-native species likely to threaten biodiversity and ecosystems in the Antarctic Peninsula region[END_REF][START_REF] Hughes | Breaking the ice: the introduction of biofouling organisms to Antarctica on vessel hulls: Vessel Biofouling In Antarctica[END_REF][START_REF] Mccarthy | -Antarctica: The final frontier for marine biological invasions[END_REF][START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF]. The development of molecular tools associated to comprehensive sampling have allowed for rapid and efficient detection of marine non-native species [START_REF] Bott | Molecular tools for detection of marine pests: Development of putative diagnostic PCR assays for the detection of significant marine pests: Asterias amurensis, Carcinus maenas, Undaria pinnatifida and Ciona intestinalis[END_REF]. In Antarctica, the Patagonian mussel Mytilus cf. platensis [START_REF] Cárdenas | -First mussel settlement observed in Antarctica reveals the potential for future invasions[END_REF] and the bryozoan Membranipora membranacea [START_REF] Avila | -Invasive marine species discovered on nonnative kelp rafts in the warmest Antarctic island[END_REF] were reported for the first time in the WAP in 2020. Both species have been categorized as "invasive non-native species likely to threaten biodiversity and ecosystems" in Antarctica [START_REF] Hughes | Invasive non-native species likely to threaten biodiversity and ecosystems in the Antarctic Peninsula region[END_REF]. Regarding green algae, some species of Chlorophyta are recorded among the most invasive marine organisms [START_REF] Williams | -A Global Review of the Distribution, Taxonomy, and Impacts of Introduced Seaweeds[END_REF] and have demonstrated to drastically affect coastal ecosystems functions (e.g., Caulerpa taxifolia introduction in Mediterranean Sea; [START_REF] Bellansantini | The influence of the introduced tropical alga Caulerpa taxifolia on the biodiversity of the Mediterranean marine biota[END_REF][START_REF] Jousson | Molecular evidence for the aquarium origin of the green alga Caulerpa taxifolia introduced to the Mediterranean Sea[END_REF]. The only non-native photosynthetic marine organism reported to be recently established in Antarctica is the green alga Ulva intestinalis [START_REF] Clayton | -New records of temperate and sub-Antarctic marine benthic macroalgae from Antarctica[END_REF]. The species was observed in highly touristic sites and closed to human settlements (e.g., scientific bases) around the SShs and the WAP, and its arrival was related to maritime transport (i.e. specimens found as biofouling on ship hull; [START_REF] Clayton | -New records of temperate and sub-Antarctic marine benthic macroalgae from Antarctica[END_REF][START_REF] Chown | -Continent-wide risk assessment for the establishment of nonindigenous species in Antarctica[END_REF][START_REF] Chown | -The changing form of Antarctic biodiversity[END_REF][START_REF] Hughes | Breaking the ice: the introduction of biofouling organisms to Antarctica on vessel hulls: Vessel Biofouling In Antarctica[END_REF]. As increasing shipping traffic augment propagule pressure of potential new colonizer [START_REF] Cárdenas | -First mussel settlement observed in Antarctica reveals the potential for future invasions[END_REF][START_REF] Hughes | Invasive non-native species likely to threaten biodiversity and ecosystems in the Antarctic Peninsula region[END_REF][START_REF] Hughes | Breaking the ice: the introduction of biofouling organisms to Antarctica on vessel hulls: Vessel Biofouling In Antarctica[END_REF][START_REF] Lee | Quantifying the propagule load associated with the construction of an Antarctic research station[END_REF], being able to detect early arrival of non-native species and to monitor their possible settlement and distribution range extension will rely on a comprehensive sampling design associated with long-term monitoring and available molecular data [START_REF] Mccarthy | -Antarctica: The final frontier for marine biological invasions[END_REF][START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF]. However, apart from [START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF] effort to resume the current state of knowledge about Antarctic seaweed diversity and distribution, long-term data monitoring is still lacking for these taxa at regional scale [START_REF] De Broyer | -How many species in the Southern Ocean? Towards a dynamic inventory of the Antarctic marine species. Deep Sea Research. Part II Topicals Studies in Oceanography, Census of Antarctic Marine Life: Diversity and Change in the Southern Ocean Ecosystems[END_REF][START_REF] Grant | Barcoding Antarctic Biodiversity: current status and the CAML initiative, a case study of marine invertebrates[END_REF].

We propose to revisit Antarctic green macroalgae diversity using two molecular markers regularly used for species determination and barcoding in Chlorophyta (i.e., the plastid genes tufA and rbcL; [START_REF] Leliaert | -Phylogeny and Molecular Evolution of the Green Algae[END_REF][START_REF] Lewis | -Unearthing the molecular phylodiversity of desert soil green algae (Chlorophyta)[END_REF][START_REF] Pröschold | Systematics of the green algae: conflict of classic and modern approaches[END_REF]. Our survey encompasses a wide area (i.e., more than 450 km) from the SShs to the center part of the WAP. The present work is part of an ongoing effort to monitor the benthic Antarctic flora, a group of taxa under increasing threat in the region due to the acceleration of climate change and intensification of anthropogenic activities. morphotypes (e.g., presenting noticeable variations in thallus shape, color or thickness and elasticity) were collected. All specimens were pressed as vouchers after removing a small portion of the thallus that was stored in silica gel for subsequent DNA analysis. Voucher specimens are housed in the herbarium of the Universidad Austral de Chile and available on request. All voucher specimens were identified, to the lowest possible taxonomic level, on the basis of morphological criteria using floristic keys and species list available for the region (Ramirez, 2010;Pellizarri et al., 2017;[START_REF] Wiencke | Antarctic Seaweeds[END_REF].

MATERIALS AND METHODS

Sampling

DNA extraction, PCR amplification and sequencing -For each specimen stored in the herbarium, a fraction corresponding to some 30 mm² of dried tissue was milled in a Mini-BeadBeater 24 (BioSpec Products, Inc. Bortlesville, USA) and DNA extraction was performed with an E.Z.N.A tissue DNA kit (Omega Bio-tek, Inc. Georgia, USA) following the manufacturer instructions.

A fragment of the plastid gene tufA, encoding for protein synthesis elongation factor Tu (EF-Tu), was amplified in all samples. This gene, well conserved in a wide variety of photosynthetic species, allows for reliable plant and green algae species determination [START_REF] Fama | -Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene[END_REF]Saunders & Kucera, 2010) and has been largely used to infer green macroalgae phylogeny [START_REF] Leliaert | -Phylogeny and Molecular Evolution of the Green Algae[END_REF]. Amplification of tufA was realized using the primers (TufAgf4: 5TGAAACAGAAMAWAWCGTCATTATGC-3 and TufAR: 5CCTTCNCGAATMGCRAAWCGC-3) developed by [START_REF] Fama | -Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene[END_REF] following the published protocol.

For a subsampling of green algae specimens (i.e. one or a few specimens per distinct genetic entities detected with the gene tufA), the plastid gene rbcL coding for the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase was amplified. The primers GrbcLnF (5' GCTGGWGTAAAAGATTAYCG 3') and GrbcLR (5' TCACGCCAACGCATRAASGG 3') developed by Saunders & Kucera (2010) were used and PCR reaction mix and program follow the protocol of [START_REF] Pirian | -Molecular and morphological characterisation of Ulva chaugulii, U. paschima and U. ohnoi (Ulvophyceae) from the Persian Gulf, Iran[END_REF].

All PCR reactions were performed in a Perkin Elmer Gene Amp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, USA). PCR products were purified using the commercial kit UltraCleanTM (MO BIO Laboratories, Carlsbad, USA). Quality and concentration of purified PCR products were verified by electrophoresis on 2% agarose gel dyed with GelRed™ (Biotium Inc, Hayward, USA). Sequencing was performed in AUSTRAL-omics Core-Facility (Universidad Austral de Chile, Chile) using a ABI PRISM®310 Genetic Analyzer (Applied Biosystems, Foster City, USA).

Data analyses -Sequences were edited using Chromas v.2.33 [START_REF] Mccarthy | Chromas, Version 1.41[END_REF], and aligned using MEGA v.5 [START_REF] Tamura | -MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Molecular species identification was performed using the basic local alignment search tool (BLAST) from NCBI [START_REF] Altschul | -3.1 Sequence searches-challenges[END_REF] and comparing the sequences obtained in this study with those available in GenBank. Given the scarcity of available sequences, a threshold of 95% identity was used as a cut-off value to record a match [START_REF] Manghisi | -DNA Barcoding Sheds Light on Novel Records in the Tunisian Red Algal Flora[END_REF].

RESULTS

A total of 122 specimens of Chlorophyta were sampled between the high intertidal down to a depth of 30 m in the sampling area: 34 in PRAT, 22 in KGI, 15 in OHI, 34 in GGV and 17 in MAR. Because of low quality and/or quantity of DNA extracted for some specimens, tufA sequences were obtained only for 93 specimens, representing 76 % of the samples. Among these 93 sequences, four were contaminated by the bacteria Granulosicoccus antarcticus [START_REF] Lee | -Granulosicoccaceae fam. nov., to include Granulosicoccus antarcticus gen. nov., sp. nov., a non-phototrophic, obligately aerobic chemoheterotroph in the order Chromatiales, isolated from Antarctic seawater[END_REF] The rbcL sequences were obtained for only half of tested specimen (N=15) belonging to seven putative species out of the twelve detected with the tufA gene (Suppl Mat 2). No rbcL PCR products were obtained for Monostroma hariotii, even after testing amplification using the DNA of the 37 specimens available. Among the 15 rbcL obtained sequences, 11 were congruent with tufA sequences identification (Suppl Mat 1). The remaining four sequences were identified as Ulva sp. A-GW and congruent with morphological identification but tufA gene sequences were lacking for these specimens. All sequences were deposited in the public depository (GenBank NCBI Public Database; see Suppl Mat 1 and 2). Following the classification of Guiry & Guiry (2019), taxonomic status regarding species-specific results provided by tufA and rbcL datasets are registered below. Reported distribution also follows Guiry & Guiry (2019). Acrosiphonia arcta has previously been reported in Antarctic waters (Ramirez, 2010), mainly under the name Spongomorpha arcta [START_REF] Papenfuss | Catalogue and bibliography of Antarctic and Subantarctic benthic marine algae[END_REF]Pellizarri et al., 2017) considered as synonymous for this species (Guiry&Guiry, 2019). We provide here the first genetic data for A. arcta in the southern part of its area of distribution (i.e., GGV in the WAP), and confirm its amphipolar distribution (Saunders & Kucera, 2010;VanOppen et al., 1993, Figure 2). [START_REF] Medeiros | Macroalgae diversity of Admiralty Bay, King George Island, Antarctic Peninsula based on DNA barcoding and other molecular markers[END_REF] and complementing morphological characters with molecular markers, [START_REF] Pellizzari | Diversity and spatial distribution of seaweeds in the South Shetland Islands, Antarctica: an updated database for environmental monitoring under climate change scenarios[END_REF] reported the presence of Protomonostroma rosulatum in the SShs instead of P. undulatum, as described in the early study of [START_REF] Vinogradova | Ad flora chlorophytorum ex Antarctide[END_REF]. However, since closest matches obtained for both genes for our sequences were with Protomonostroma sp. A-GW we decided to use this last name for specimens sampled in the present study until further taxonomic work. Urospora sp. 1 penicilliformis (SShs: PRAT, WAP: GGV, MAR) Reported distribution of Urospora penicilliformis: Worldwide except tropical waters. Exact match was found with Urospora sp. 1 penicilliformis tufA sequence from Nome, Alaska (GenBank Access Number MH571163, Suppl Mat 1). Another close match was found (99.48%, GenBank Access Number HQ610440, Suppl Mat 1) with Urospora sp. 1 penicilliformis tufA sequence from Canada. Exact match was found for the rbcL gene with Urospora sp. 1 penicilliformis sequence from USA, Maine (GenBank Access Number HQ603674, Suppl Mat 2). Type locality for Urospora penicilliformis is located in the northern hemisphere, probably in Germany (Guiry & Guiry, 2019), but the species has previously been reported (based on morphological character) in the southern hemisphere along the Chilean and Argentinean coasts (Boraso de [START_REF] Boraso De | -Chlorophyta marinas de la Argentina[END_REF][START_REF] Boraso De Zaixso | Elementos para el estudio de las macroalgas de Argentina[END_REF]Ramirez y Santelices, 1991), Antarctica and Sub-Antarctic Islands [START_REF] Papenfuss | Catalogue and bibliography of Antarctic and Subantarctic benthic marine algae[END_REF][START_REF] Mystikou | Seaweed biodiversity in the southwestern Antarctic Peninsula: surveying macroalgal community composition in the Adelaide Island/Marguerite Bay region over a 35-year time span[END_REF][START_REF] Wiencke | Antarctic Seaweeds[END_REF], Australia and New Zealand [START_REF] Broady | -Phylum Chlorophyta and Charophyta: green algae. In: New Zealand inventory of biodiversity v3. Kingdoms Bacteria[END_REF][START_REF] Womersley | The marine benthic flora of southern Australia[END_REF]. Amphipolar distribution of Urospora sp 1 penicilliformis is supported by molecular data (Alaska: Bringloe et 2017). No rbcL sequences were obtained for this specie in the present study. Since our samples were first determined as M. hariotii based on morphological characters and due to the fact that M. hariotii has been reported as an emblematic specie of the Antarctic and SubAntartic waters [START_REF] Wiencke | Antarctic Seaweeds[END_REF] while M. angicava has only been reported in the northern hemisphere, we decided to retain the name M. hariotii for specimens sequenced in the present study. Monostroma hariotii has been reported as common in the Falklands, Kerguelen and Macquarie Islands [START_REF] Wiencke | Antarctic Seaweeds[END_REF], South Orkney Islands [START_REF] Wiencke | Antarctic Seaweeds[END_REF], South Shetlands Islands including King George Island [START_REF] Guillemin | -Marine epiphytic diatoms from the shallow sublittoral zone in Potter Cove, King George Island, Antarctica[END_REF]Pellizzarri et al., 2017;[START_REF] Quartino | -Biological and environmental characterization of marine macroalgal assemblages in Potter Cove, South Shetland Islands, Antarctica[END_REF][START_REF] Wiencke | Antarctic Seaweeds[END_REF], Wilkes Land [START_REF] Runcie | Photosynthesis of marine macroalgae in ice-covered and ice-free environments in East Antarctica[END_REF] and the Antarctic Peninsula [START_REF] Amsler | Potential chemical defenses against diatom fouling in Antarctic macroalgae[END_REF][START_REF] Lamb | -Benthic marine algae of the Antarctic Peninsula[END_REF][START_REF] Mystikou | Seaweed biodiversity in the southwestern Antarctic Peninsula: surveying macroalgal community composition in the Adelaide Island/Marguerite Bay region over a 35-year time span[END_REF][START_REF] Peters | -A comparative analysis of the nutritional and elemental composition of macroalgae from the western Antarctic Peninsula[END_REF].

CHLOROPHYTA, Ulotrichales, Ulotrichaceae

CHLOROPHYTA, Ulvales, Ulvaceae

Ulva sp. A-GW, (WAP: GGV, MAR). Reported distribution: Unknown. Both genes tufA and rbcL confirmed the assignation of our samples to the species Ulva sp. A-GW with exact matches with sequences from King George Island, SShs (100%, GenBank Access Number MG646368 and MG711515 for tufA and rbcL genes, respectively, Suppl. Mat. 1 and 2).

Close matches were also observed with tufA sequences from British Columbia, Canada (98.83%, GenBank Access Number KM254999) and Nome, Alaska (98.61%; GenBank Access Number MF124264; Suppl Mat 1) suggesting that Ulva sp. A-GW displays an amphipolar distribution (Figure 2). Our specimens were sampled in the southern part of the WAP (e.g. Gerlache Strait and Marguerite Bay), about 250 km from King George Island, suggesting an extensive distribution of Ulva sp. A-GW, at least in the Western Antarctic.

Unknown specimen of Ulvales (WAP: GGV). Reported distribution: Unknown. One sample formerly identified as Urospora penicilliformis show a close match with a specimen of Ulvales isolated from Mastocarpus stellatus in England for the tufA gene (95.98%, Ulvales sp. 6 BER, GenBank Access Number EF595318, Suppl Mat 1). Since the rbcL gene failed to amplify, no accurate identification is available for this sample. The sequence obtained in the present study could correspond to an epiphytic Ulvales specimen living on U. penicilliformis.

CHLOROPHYTA, Ulvales, Ulvellaceae

Ulvella islandica R. Nielsen & K. Gunnarsson (SShs: PRAT). Reported distribution:

North Europe (Iceland). After sequencing one specimen formerly identified as Monostroma hariotii using morphological characters, molecular data provided unexpected close match with Ulvella islandica tufA sequence from Iceland (98.12%, GenBank Access Number KF444924, Suppl Mat 1). It is probable that the sequence obtained in the present study corresponds to an epiphytic Ulvella stage living on M. hariotii thallus. No rbcL sequence was obtained for this specimen. Ulvella islandica has been recently described in Icelandic waters (Nielsen et al. 2014) but has never been reported in Antarctic waters. The tufA sequence obtained in the present study was less related to other Ulvella sequences deposited in GenBank (Ulvella reticulata, 96.54%, GenBank Access Number JQ303009; Ulvella viridis, 95.61%, GenBank Access Number EF595286; Ulvella leptochaete, 95,07%, GenBank Access Number JQ303013, Suppl Mat 1), all species reported in Antarctica [START_REF] Mystikou | Seaweed biodiversity in the southwestern Antarctic Peninsula: surveying macroalgal community composition in the Adelaide Island/Marguerite Bay region over a 35-year time span[END_REF][START_REF] Pellizzari | Diversity and spatial distribution of seaweeds in the South Shetland Islands, Antarctica: an updated database for environmental monitoring under climate change scenarios[END_REF]. This could represent the first evidence of an amphipolar distribution for Ulvella islandica, but caution should be taken as our study relies upon one single specimen sampled in PRAT.

CHLOROPHYTA, Trebouxiophyceae, Prasiolales

Prasiola crispa (Lightfoot) Kützing (WAP: OHI, GGV). Reported distribution:

Worldwide. Two of the three specimens sequenced for tufA showed exact matches with GenBank sequence of Prasiola crispa strain n°43 from King George Island (100%, GenBank Access Number KF993450, Suppl Mat 1) while the other one exactly matched a sequence of P. crispa from Svalbard (100%, GenBank Access Number LN877821, Suppl Mat 1). rbcL gene confirms Prasiola crispa identification and an exact match was encountered with a Canadian specimen from British Columbia (100%, GenBank Access Number KR017748, Suppl Mat 2). Our findings are congruent with previous works reporting the presence of the species in Antarctica and its amphipolar distribution pattern (Garrido-Benavant et al., 2017;Moniz et al., 2012).

Prasiola crispa subsp. antarctica (Kützing) Knebel (WAP: GGV). Reported distribution:

Antarctic and the subAntarctic islands (Macquarie Island -South Georgia -SShs -Antarctic Peninsula) -South America (Chile -Argentina). Close match was found for a single specimen formerly identified as Prasiola sp. with P. antarctica strain P31 from the SShs for the tufA gene (99.31%, GenBank Access Number KF993447, Suppl Mat 1). Exact match for rbcL sequence was found with the same specimen of P. antarctica strain P31 from the SShs (100%, GenBank Access Number JQ669712, Suppl Mat 2). Moniz et al. (2012) proposed the resurrection of P. antarctica as a true species. However this decision has not yet been approved and P. antarctica is still considered as a synonym of Prasiola crispa in AlgaeBase. Our sequence was thus named Prasiola crispa subsp. antarctica, after AlgaeBase nomenclature. Our work expands the distribution of Prasiola crispa subsp. antarctica from the SShs (Moniz et al., 2012) New Zealand -Argentina. One specimen, formerly identified as Blidingia minima using morphological characters, showed close match with Rosenvingiella radicans from Norway for the tufA gene (98.02%, GenBank Access Number LN877834, Suppl Mat 1). Rosenvingiella radicans has only been described in the Northern hemisphere. Another species of Rosenvingiella, R. simplex, has been described along the coasts of King George Island [START_REF] Vinogradova | Ad flora chlorophytorum ex Antarctide[END_REF]. To the extent of our knowledge, no tufA sequence representing this species has been deposited in public repositories, while 8 rbcL sequences are available from Norway (GenBank Access Number LN877833 -LN877833 -AY694199:AY694204, Heesch et al., 2016). Unfortunately, rbcL gene failed to amplify in the present study limiting further identification.

DISCUSSION AND CONCLUSION

New records of Chlorophyta in Antarctic waters

Molecular data obtained for 86 specimens of our 122 Chlorophyta samples allowed detecting twelve species including three new reports (Rosenvingiella radicans, Urospora wormskioldii and Ulvella islandica) in the SShs and WAP area. Urospora wormskioldii was previously reported in the northern hemisphere close to the polar circle, along the coasts of Greenland, Canada, Europe and East Russia (Guiry & Guiry, 2019; Figure 2), Ulvella islandica in Iceland and Rosenvingiella radicans at mid-high latitudes in both hemispheres (Guiry & Guiry, 2019). A cosmopolitan distribution (except in the tropics, Guiry & Guiry, 2019) has been reported for Urospora penicilliformis, a specie considered as common in the intertidal zone and reported in the SShs and WAP since first register in the middle of the 20th century [START_REF] Lamb | -Benthic marine algae of the Antarctic Peninsula[END_REF][START_REF] Mystikou | Seaweed biodiversity in the southwestern Antarctic Peninsula: surveying macroalgal community composition in the Adelaide Island/Marguerite Bay region over a 35-year time span[END_REF][START_REF] Papenfuss | Catalogue and bibliography of Antarctic and Subantarctic benthic marine algae[END_REF][START_REF] Roleda | -Sensitivity of Antarctic Urospora penicilliformis (Ulotrichales, Chlorophyta) to ultraviolet radiation is life-stage dependent[END_REF][START_REF] Wiencke | Antarctic Seaweeds[END_REF]. We reported here, for the first time, the presence of a second specie of Urospora, U. wormskioldii, in Antarctica.

The present work improves the Chlorophyta genetic database in a region within which only a few green macroalgae have been sequenced (Ulvophyceae: Monostroma grevillei, [START_REF] Pellizzari | Diversity and spatial distribution of seaweeds in the South Shetland Islands, Antarctica: an updated database for environmental monitoring under climate change scenarios[END_REF]Ulva sp.: Khan et al., 2017;Trebouxiophyceae: Prasiola crispa and Prasiola antarctica: Garrido-Benavant et al. 2017;Moniz et al. 2012). Lack of molecular data, especially of sequences available in public repositories for comparison in barcoding studies, has been identified as a clear limitation for studies focused on Antarctic algae (Dubrasquet et al., 2018). Even if only a few species of green algae are reported in Antarctica, the use of morphological characters without confirmation by molecular data could lead to confusion and inaccuracy in assessing marine flora diversity. In the present study, even if only a few sequences of Antarctic green algae were available in public repositories, identification match with GenBank reference sequences were obtained with at least one of the two genetic markers (i.e. tufA and rbcL genes) for each putative species, most of them with sequences from specimens sampled in the northern hemisphere. These new tufA and rbcL data, including sequences of common intertidal species such as Acrosiphonia arcta, Monostroma hariotii, Ulva sp. A-GW and Urospora penicilliformis could help in Antarctic algae diversity longterm monitoring. However, as in other polar areas (i.e. Alaska, Bringloe et al. 2019), assessing the current state of marine flora diversity in Antarctica will imply important sampling effort in order to include specimens from other non-glaciated coasts, such as East Antarctic coasts located between 45°E and 160°E [START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF], to complete the information already obtained for the SShs and the WAP [START_REF] Papenfuss | Catalogue and bibliography of Antarctic and Subantarctic benthic marine algae[END_REF]Ramirez, 2010;[START_REF] Wiencke | Antarctic Seaweeds[END_REF][START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF]present study). Our sampling strategy was limited by logistics of the Antarctic campaigns and sampling restrictions: sampling effort happening only during summer season, one sampling event in each region and scuba diving down to 30m (e.g. the emblematic species Lambia antarctica, that generally live at greater depth as reported by [START_REF] Wiencke | -Biogeographic Atlas of the Southern Ocean. Chapter 5.1. Macroalgae[END_REF], was not sampled in any of our five sites). However, our effort still allowed us to sample and sequenced half of the reported species in the SShs and WAP (12 over 24, Pellizzarri et al., 2017).

Various Antarctic green algae are amphipolar species

The use of molecular data allows a better understanding of marine flora diversity but also to better define species biogeographic limits and study their evolutionary history. Recent studies have focused on current diversity and distribution pattern of red [START_REF] Billard | -Deep genetic divergence between austral populations of the red alga Gigartina skottsbergii reveals a cryptic species endemic to the Antarctic continent[END_REF][START_REF] Dubrasquet | -Molecular-Assisted Revision of Red Macroalgal Diversity and Distribution along the Western Antarctic Peninsula and South Shetland Islands[END_REF][START_REF] Guillemin | -Comparative phylogeography of six red algae along the Antarctic Peninsula: extreme genetic depletion linked to historical bottlenecks and recent expansion[END_REF][START_REF] Ocaranza-Barrera | -Molecular divergence between Iridaea cordata (Turner) Bory de Saint-Vincent from the Antarctic Peninsula and the Magellan Region[END_REF] and brown [START_REF] Peters | -Phylogeny and historical ecology of the Desmarestiaceae (Phaeophyceae) support a southern hemisphere origin[END_REF][START_REF] Peters | -The phylogenetic position of the subantarctic marine macroalga Desmarestia chordalis (Phaeophyceae) inferred from nuclear ribosomal ITS sequences[END_REF] Antarctic algae. Cryptic species have been found in several wellknown and widely distributed red algae [START_REF] Billard | -Deep genetic divergence between austral populations of the red alga Gigartina skottsbergii reveals a cryptic species endemic to the Antarctic continent[END_REF][START_REF] Dubrasquet | -Molecular-Assisted Revision of Red Macroalgal Diversity and Distribution along the Western Antarctic Peninsula and South Shetland Islands[END_REF][START_REF] Guillemin | -Comparative phylogeography of six red algae along the Antarctic Peninsula: extreme genetic depletion linked to historical bottlenecks and recent expansion[END_REF] and in terrestrial green algae (DeWever et al., 2009), underlying the limitation of taxonomic knowledge for these taxa.

For Antarctic species, as for the canopy forming brown algae Desmarestia spp. and the common red algae Gigartina skottsbergii, divergence from species living outside of the Antarctic waters has been estimated to date back some 10 Million years (Mya) [START_REF] Billard | -Deep genetic divergence between austral populations of the red alga Gigartina skottsbergii reveals a cryptic species endemic to the Antarctic continent[END_REF][START_REF] Peters | -Phylogeny and historical ecology of the Desmarestiaceae (Phaeophyceae) support a southern hemisphere origin[END_REF]. As a result of long-time isolation from the rest of the marine realms, red and brown Antarctic algae display a high percentage of endemic species nowadays (36% and 44% respectively) and clear adaptations to Antarctic marine environment. However, some cold-water species of Desmarestia (i.e Desmarestia aculeata, D. viridis/confervoides, [START_REF] Peters | -Phylogeny and historical ecology of the Desmarestiaceae (Phaeophyceae) support a southern hemisphere origin[END_REF]D. viridis/willii, VanOppen et al., 1993) have been reported in both cold Arctic and Antarctic waters (note that D. confervoides is considered as synonymous of D. willii, Guiry & Guiry. 2019). For Antarctic green algae, a much lower percentage of endemic species has been recorded (18%, [START_REF] Wiencke | Antarctic Seaweeds[END_REF] and amphipolar distribution has been observed for the common intertidal species Acrosiphonia arcta (VanOppen et al., 1993).

For both D. viridis/willii and A. arcta, amphipolar distribution has been related to recurrent equator-barrier crossing during the cooling temperatures events of the Pleistocene (VanOppen et al., 1993). The ability of early life stages to survive extreme temperatures is crucial when considering a possible connection from pole to pole and phytogeographical patterns and endemism levels are shaped by this physiological requirement (Bartsch et al., 2012 and references therein). Gametophyte stages of D. viridis/willii and A. arcta present great tolerance to warm temperatures (i.e. survival up to 26-27°C for D. viridis/willii and at least up to 25°C for A. arcta; [START_REF] Peters | Temperature responses of disjunct temperate brown algae indicate long-distance dispersal of microthalli across the tropics[END_REF]VanOppen et al, 1993). These two species could have survived the passage of the tropics through deep-water dispersion of gametophyte stages. Early life stages of several Antarctic marine green algae as A. arcta, Ulva sp. and U.

penicilliformis have been shown to present better tolerance to high temperatures (upper survival temperature above 20°C) than endemic red or brown algae (upper survival temperature between 11°C and 19°C, Wiencke & Dieck, 1990). In general, green algae propagules have been shown to support long dark periods (e.g., Ulva flexulosa; [START_REF] Imchen | -Recruitment Potential of a Green Alga Ulva flexuosa Wulfen Dark Preserved Zoospore and Its Development[END_REF] and to be able to travel over very long distances on oceanic currents (more than hundreds of kilometers; [START_REF] Watanabe | -Dispersal potential of the invasive green alga Codium fragile ssp. fragile[END_REF] attached to rafting algae [START_REF] Arroyo | -The role of drifting algae for marine biodiversity[END_REF][START_REF] Macaya | -Seaweeds Phylogeography. Chapter 4. Float and Raft: Role of Buoyant Seaweeds in the Phylogeography and Genetic Structure of Non-buoyant Associated Flora[END_REF]Saunders, 2014). This could explain in part their success as invasive species (e.g Caulerpa taxifolia: [START_REF] Arnaud-Haond | Entangled fates of holobiont genomes during invasion: nested bacterial and host diversities in Caulerpa taxifolia[END_REF][START_REF] Fama | -Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene[END_REF][START_REF] Bellansantini | The influence of the introduced tropical alga Caulerpa taxifolia on the biodiversity of the Mediterranean marine biota[END_REF][START_REF] Smith | Fragmentation as a strategy for Caulerpa species: Fates of fragments and implications for management of an invasive weed[END_REF]; Codium fragile sp fragile: [START_REF] Watanabe | -Dispersal potential of the invasive green alga Codium fragile ssp. fragile[END_REF] or the high number of species with a reported amphipolar distribution. A recent study on evolutionary history of the lichen-associated green algae Prasiola crispa complex species proposed a combined theory of vicariance events associated with long distance deep-water dispersal across the tropics during the Pleistocene for explaining their disjoint distribution in both polar areas (Garrido-Benavant et al., 2017). Our findings confirm the existence of an amphipolar distribution for A. arcta, P. crispa, U. penicilliformis, U. wormskioldii, Ulva sp.

A-GW and Ulvella islandica and show that amphipolar distribution seems to be much more common in Antarctic green than red or brown algae. It could explain in part the low level of endemism found in Antarctica for these studied taxa [START_REF] Wiencke | Antarctic Seaweeds[END_REF].

Conclusion

Studies of species diversity including genetic data provide key information for correct assessment of flora and fauna diversity in Antarctica, an area still difficult to access [START_REF] Dubrasquet | -Molecular-Assisted Revision of Red Macroalgal Diversity and Distribution along the Western Antarctic Peninsula and South Shetland Islands[END_REF][START_REF] De Broyer | -How many species in the Southern Ocean? Towards a dynamic inventory of the Antarctic marine species. Deep Sea Research. Part II Topicals Studies in Oceanography, Census of Antarctic Marine Life: Diversity and Change in the Southern Ocean Ecosystems[END_REF][START_REF] Grant | Barcoding Antarctic Biodiversity: current status and the CAML initiative, a case study of marine invertebrates[END_REF][START_REF] Fučíková | -New phylogenetic hypotheses for the core Chlorophyta based on chloroplast sequence data[END_REF]. Fast environmental changes have been reported in Antarctica, especially in the SShs and the WAP, leading to increasing pressures and threats over the Antarctic biota [START_REF] Chown | -The changing form of Antarctic biodiversity[END_REF]. Among them, introduction of non -native species associated to human activities such as scientific research and tourism have been reported as an important threat (Broady et al., 1994;[START_REF] Mccarthy | -Antarctica: The final frontier for marine biological invasions[END_REF][START_REF] Olech | Human impact on terrestrial ecosystems in west Antarctica[END_REF][START_REF] Radulovici | -DNA Barcodes for Marine Biodiversity: Moving Fast Forward?[END_REF], Cardenas et al. 2020).

However, basic information is still lacking that could allow to properly monitor the timing and magnitude of these arrivals. Quick detection of alien species settlement in Antarctic waters could help building environmental recommendation for shipping, including tourism and fishing activities. As these organisms present great dispersal potential and includes several potential invaders, availability of genetic sequences in public depository for species commonly found in the Western Antarctic Peninsula and the South Shetland Islands will help to monitor the state of green Antarctic flora. (Guiry & Guiry, 2019)

JAMES ROSS ISLAND

Weddell Sea

A N T A R C T I C P E N I N S U L A

mm

Urospora wormskioldii

Existing genetic data New genetic data

  -Sampling was realized during austral summers between 2011 and 2014 within the framework of four campaigns organized by the Chilean Antarctic Institute (INACH). Five areas were sampled (Figure 1), two located in the SShs (near the Chilean Capitán Arturo Prat base in Greenwich Island and at Bahia Fildes in King George Island, hereafter referred as PRAT and KGI, respectively) and three areas along the Northern and Central part of the WAP (near the Chilean O'Higgins Antarctic base, noted OHI; in Paradise Bay, near the Chilean Presidente Gabriel González Videla Antarctic base, noted GGV and in Marguerite Bay, noted MAR). In all areas, intertidal samplings were conducted during diurnal low tide hours while subtidal samples were collected by SCUBA diving. Specimens showing different

  and three by the diatom Seminavis robusta D.B.Danielidis & D.G.Mann and removed from the dataset. The remaining 86 sequences obtained for green macroalgae belonged to Ulvophyceae (69 specimens of Ulotrichales and 12 specimens of Ulvales) and Trebouxiophyceae (5 specimens belonging to Prasiolales). Twelve distinct putative species were detected in the present study using tufA molecular dataset: nine are part of the Ulvophyceae class and three of the Trebouxiophyceae class (Suppl Mat 1). In order to confirm the species assignation based on the tufA gene, the rbcL gene was amplified in a subsample of randomly selected specimens (N=31 in total) belonging to each putative species.

  Caledonia. Closest match (98,31%, GenBank Access Number HQ610211, Suppl Mat 1) with Antarctic tufA sequences was a Canadian specimen of Acrosiphonia arcta from British Columbia. No rbcL sequences were obtained for this species.

  ). No close match was found in GenBank for the tufA sequences obtained in the present work. Close match was found for rbcL sequences of specimens sampled in the Gerlache Strait (formerly identified as Urospora penicilliformis using morphological characters in the present study) with the Capsosiphon fulvescens plastid complete genome (98.53%, GenBank Access Number NC_039920, Suppl Mat 2). Capsosiphon fulvescens has never been reported in the Antarctic. However, using morphological identification combined to the information from various nuclear markers (18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2 and 50-28S rRNA gene),[START_REF] Mystikou | Seaweed biodiversity in the southwestern Antarctic Peninsula: surveying macroalgal community composition in the Adelaide Island/Marguerite Bay region over a 35-year time span[END_REF] reported the presence of Capsosiphon groelandicus in the WAP (i.e Adelaide Island in Marguerite Bay). Since species identification could not be clarified with the help of our two molecular markers, we choose to keep the name Capsosiphon sp. for the specimens sampled in the present study until further taxonomic work. Protomonostroma sp. A-GW (SShs: PRAT). Reported distribution: P. undulatum has been reported in Europe (UK -Norway -Germany -Faroe Islands -Iceland -Greenland -Denmark) -USA (Maine -Alaska) -Canada (British Columbia -New Brunswick) -North Asia (Japan -China -Korea -Kamchatka -East Russia) -Argentina. P. rosulatum Vinogradova, has only been reported in the South Shetland Islands. Closest match for tufA gene (99.87%, GenBank Access Number MG646367, Suppl Mat 1) was found with Protomonostroma sp. A-GW from King George Island. Exact match with rbcL sequences of Protomonostroma sp. A-GW from King George Island was observed (100%, GenBank Access Number MG711514, Suppl Mat 2). Based on previous reports

  al. 2019; British Columbia: Saunders & Kucera, 2010; Antarctic and subAntarctic Islands: the present study, Figure 2). Urospora wormskioldii (Mertens) Rosenvinge (WAP: OHI). Reported distribution: Arctic (Canada -Svalbard -Greenland -Iceland & Faroe Islands) -North Europe (Germany -Denmark -Brittany -Baltic Sea -Norway -Spitzberg) -North America (both Pacific and Atlantic coasts down to Mexico) -North Asia (China -East Russia -Kamchatka). Closest matches for three specimens formerly identified as U. penicilliformis based on morphological characters were observed with sequences of Urospora wormskioldiifrom British Columbia, Canada for both tufA and rbcL genes (99.87%, GenBank Access Number HQ610441 and HQ603676 for tufA and rbcL, respectively, Suppl Mat 1 and 2). The present molecular data represent the first report of a second Urospora species in Antarctic waters, underlying the unknown amphipolar distribution pattern of U. wormskioldii(Lindstrom & Hanic, 2005, Figure 2). Islands). Exact match for tufA gene was observed with sequence of a specimen named Monostroma angicava from King Georges Island (100%, GenBank Access Number MG646366, Suppl Mat 1). Comparison with other sequences of specimens of Monostroma available in public repositories showed a lower percentage of similarity (e.g. 92.60% of similarity with Monostroma grevillei sp. 1 from Canada, GenBank Access Number HQ610257). The species Monostroma grevillei sp. 1 has, however, been reported, identified by molecular approaches, in the South Shetland Islands (Pellizarri et al.

  down to the Gerlache Strait. Rosenvingiella radicans (Kützing) Rindi, L.McIvor & Guiry (SShs: PRAT). Reported distribution: North Europe (Britain -Ireland -Baltic Sea -France -Faroe Island -Spain) -North America (California -Washington) -Arctic (White Sea) -Australia &

FIGURES LEGENDFigure 1 :

 1 FIGURES LEGEND

Figure 2 :

 2 Figure 2: Amphipolar distribution of four species of Chlorophyta detected in the SShs and/or

  . Genetic data (i.e., tufA) available for the species are indicated with stars: red stars, present study; black stars, already published in GenBankNCBI Database. All illustrations were kindly realized by Enzo Mardones, based on

					B SOUTH SHETLAND r a n s f i e l d KGI ISLAND PRAT S t r a i t KING GEORGE ISLAND DECEPTION OHI ISLAND
	photographies	available	in	www.algaebase.com S t r a i t	(for	Acrosiphonia	arcta),
			ANVERS ISLAND	e e r l a c h G
					GGV
	MAR

www.seaweedsofalaska.com (for Acrosiphonia arcta, Urospora wormskioldii, Urospora sp.1 penicilliformis, Ulva sp A-GW). Cells illustrations for Urospora wormskioldii and Urospora sp.1 penicilliformis were based on www.algaebase.com photographies available for Urospora penicilliformis and Urospora wormskioldii. Ulva sp. A-GW illustration was based on Ulva linza photography available at www.seaweedsofalaska.com/ A n t a r c t i c S o u n d

Acknowledgments

This research was supported by the Instituto Antártico Chileno (INACH) T_16-11 and RG_15-16 projects and sampling in King George Island was funded by the Project Anillo ART1101 from the ex Comisión Nacional de Investigación Cientıfíca y Tecnológica, now ANID "Agencia Nacional de Investigación y Desarrollo. Additional support came from the Centro FONDAP IDEAL no. 15150003. The authors thank J. L. Kappes, T. Heran, Y. Henriquez, and L. Vallejos for their help in the field and C. Cárdenas Quilapán for her help in the lab and Enzo Mardones for the seaweeds illustrations in Figure 2. The authors would also like to thank the Chilean Navy (especially the captain and crew of the ships Almirante Oscar Viel and Lautaro), the staff from the Chilean Army in the O'Higgins base and the Air Force of Chile (FACh) for the logistic support of our fieldwork in sub-Antarctica and Antarctica.

Supplementary table 1: Genbank (GB) Access Number for the tufA gene for green macroalgae specimens from the South Shetlands Islands and Western Antarctic Peninsula. Specie names are given following molecular assignation. Closest match with existing tufA sequence in Genbank data repository are given (percentage of similarity). References are given for closest match sequences. Specimens ID marked with * or ** correspond to subtidal samples (*sampling depth between 0-15m and ** sampling depth between 15-30m). Specimens ID written in bold correspond to samples for which rbcL sequences are available.

Specie name (AlgaeBase Current

Accepted name)