Nicolas Poirier

Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion

published or not. The documents may come

Introduction

Off-site material collected by fieldwalking surveys has long been considered a sort of 'background noise', an impediment to the detection of sites. In the last forty years, the extension of archaeologists' interest from sites to wider territories has led to a reconsideration of such off-site material, and its use as a marker for formerly cultivated areas [START_REF] Wilkinson | The definition of ancient manured zones by means of extensive sherd-sampling techniques[END_REF] .

Archaeological evidence for manuring in agriculture

While the presence of off-site material is clearly discernible, its interpretation as the product of manuring practices is based on an initial assumption that the spreading of livestock manure collected in barns was actually employed in past periods. To confirm this hypothesis, we will first focus on the identification of written and iconographic sources attesting an ancient practice of manuring. Most of the Roman authorities on agriculture recommended the use of manure to improve the quality of the soil [START_REF] Cato | Res Rusticae. -Columella, De Agricultura. -Pliny the Elder[END_REF] . These texts and recommendations on the use of manure were compiled and passed on by the agronomic literature of the Middle Ages from the 13 th century onwards. As early as the Carolingian period, the 'Capitulare de Villis' had advised farmers to spread not only manure, but also the remaining waste produced by their farms [START_REF] Delatouche | Regards sur l'agriculture aux temps carolingiens[END_REF] . Medieval iconography also attests to the practice of manuring [START_REF] Mane | L'iconographie des manuscrits du Traité d'agriculture de Pier'de Crescenzi[END_REF] . The Rustican contains an agricultural calendar illustrating this practice for the month of February: two men are spreading manure in an enclosed field where many other piles have been already dispersed 5 .

The second assumption is that domestic waste, including sherds of pottery, could actually be incorporated into the manure, whether deliberately or not. These sherds and fragments of building materials were mixed with other waste on manure piles (which were usually located near the settlement), and then spread across the fields alongside the organic material. Only the former would have survived once the organic matter had decomposed. This assumption is supported by a number of sources confirming the incorporation of household waste, including the sherds of broken pottery, in the manure spread:

-the proximity between houses and stables or manure pits, -the recommendation of this incorporation by agronomists, -and the confirmation of this practice by archaeological sources, geochemical observations and archival texts. Considering all of these arguments, we can conclude that the off-site material collected during fieldwalking surveys may indeed reflect the location of intensively cultivated areas. Research in this direction has permitted us to move on from viewing surveys only as a tool for the discovery of settlement sites, to using it a means of understanding landscape as a continuous space, where 'off-site' material can mark the influence of settlements on the surrounding area. However, the location of areas improved by manuring will only provide a minimum estimate of the effectively exploited area.

Of course, the absence of off-site material does not allow the reverse conclusion that the areas in question were not cultivated, but may open up a range of further possible explanations which may not allow a choice at this point.

Arkeotracker, a high-resolution geolocation tool for fieldwalking

The demand for a tool

The traditional method of examining a rural area is to survey it through a dense mesh of prospectors collecting visible artefacts from its surface. The identification of a site will occur when an abnormal concentration of artefacts is observed. At this point, it is possible to expand the method in order to try to delineate the contours of the site and estimate the intensity of the occupation. In most cases, it is only at this stage that the question of 'fine spatialization' of the remains comes into play [START_REF] Banning | Archaeological survey manuals in archaeological method, theory, and technique[END_REF] . This spatialization can be done by recording the objects according to pre-defined grid tiles, sampling the remains on transects [START_REF] Cherry | Landscape Archaeology as Long-Term History: Northern Keos in the Cycladic Islands[END_REF] , or by systematically pin-pointing the remains through the use of a total station or GPS. The arrival of GPS has significantly improved our capabilities for recording and the speed of their implementation [START_REF] Tripcevich | Flexibility by design: How mobile GIS meets the needs of archaeological survey[END_REF] . This employment, however, is limited to site registration and analysis. Systematic georeferencing of the data collected outside the sites has been attempted, but remains experimental, due not only to the technical complexity it induces, but also to its low profitability when we consider the time spent on the documented surface [START_REF] Kotarba | Hérault): Un exemple de l'apport des méthodes fines de lecture du sol en prospection pour la connaissance des sites de l'Age du Fer[END_REF] .

In order to overcome this barrier, it was necessary to develop a method which could address several constraints.

-Economy of time expenditure: The conventional use of a GPS employs a posteriori positioning of objects marked on the surface of the ground. While this method provides the advantage of accuracy, it is excessively expensive in time. It is hard to envisage its use outside confined sites on extensive plots where the sampling of material may result in the discovery of several hundred to several thousand artefacts per collection unit. The only solution to this problem would appear to be the immediate recording of information at the time of discovery.

-Accuracy and error probability in measurement: As E. Bannings pointed out 10 , there are many factors which can interfere with the measurement of an occupation area by a survey. These include human factors (inexperience or unequal experience within a team of prospectors), climatic influences (on the visibility of the vestiges) or specifics related to the nature of the sites (representativity of a set of artefacts). Taking the above factors into account in order to improve the detection and evaluating capacity for the discovered remains was another objective of our work.

-Cost-efficiency: As surveys are usually carried out within the framework of universities and non-profit archaeology, it was necessary to develop a technique which could be supported by a conventional research team both in Europe and, as far as possible, in countries on a lower economic level.

Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion -Ease of use: The last major constraint which was addressed by the project was complexity. With an emphasis on simplicity, a non-specialized student had to be able to take charge and implement our method with a training reduced to a few minutes. This was the framework within which we developed a smart phone application dedicated to assisting fieldwalking surveys. The idea was to take advantage of both the general availability of smart phones and the integration of GPS devices in the former which provide sufficient precision for the given objective.

The concept of the application

Our dedicated application, called Archeotracker 11 , contains the essential code of an open source application developed by O. Guillaumin as part of the Open Street Map project [START_REF]OSM Tracker[END_REF] . The initial application, OSMTracker has been adapted for archaeological use by Emile Hautefeuille.

Its operating principle is very simple. Each prospector must be equipped with a telephone using Android OS, on which the application was previously installed. The phone is worn on the arm in a waterproof case.

The application works without network contact, and it is recommended to set the phone to airplane mode in order to save energy.

Managing the collected data is simple. The application allows a prospector to record both his tracks and the artefacts he has observed and collected at the same time. Once a zone to be surveyed has been defined, the prospector starts a track which can be named with a dedicated registration code.

As soon as the track is opened, the application strictly records all the movements of the prospector at the rate of one point every second. This makes it possible to measure the speed of movement, and thereby the thoroughness of his collecting. When the track is opened, the application presents a simple interface which enables the prospector to record the artefacts according to major categories. Thus, when a pottery sherd is found, pressing the appropriate button of the screen makes it possible to record its location with a margin of error ranging from 2 to 5 m, depending on the GPS chip model fitted to the smart phone.

As it is theoretically possible to identify each artefact with its own registration number, the emphasis here is to register the objects according to major categories. And as the survey is carried out in parallel strips in the conventional manner, each prospector can be equipped with the tool. Recording is limited to simply pressing the screen of the phone; the operation has no impact in terms of time. It is also possible to add a written note to comment on a given object.

When the survey line is completed, the recording mode is put on hold, to resume at the start of the next line. When a site is discovered, it is of course possible to record it on a specific track file which has its own registration code. Whatever the method used, it is then possible to geolocate the set of objects attached to the site. Each prospector can observe the spatial distribution of his discoveries in real time. This allows him to review places he has already passed through in a Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion random search of artefacts within a site. The tool can also be used to correct a trajectory when the gaps between prospectors are relevant.

Once a site has been prospected, it is possible to return to the prospecting zone file and resume the original line at the point where it was left.

When a day of surveying is completed, it is only a minor effort to retrieve the data for aggregation on a GIS. The GPS tracks recorded in each phone can be transmitted via Bluetooth connection to the team leader's phone. It can then merge the traces of the day into a single file, exportable in GPX format for use under GIS. Below, we will demonstrate how this data can be used to refine the interpretation of the results.

The main factors of input

Validity of the corpus of measured data

As the registration of artefacts relies on students with variable field experience, our first observation concerning the results generated by the application is that our operating choices are, by and large, relevant.

When we consider the 124 collection units which benefited from the use of this application in 2015 and 2016, we can observe a good match between the total number of artefacts registered via the application and the count of the collected objects at the end of the field (Fig. 1). Indeed, over 75 % of the collection units display a difference of less than 25 % of this total number of artefacts, and nearly 90 % of collection units have a difference of less than 50 %. These observations are generally reassuring with regard to the users' handling of the application. The few deviations observed probably correspond to involuntary "clicks" related to the tactile character of the interface.

Accuracy in the identification of sites

The procedure which we followed in our field operations testing the feasibility of using the Archeotracker application consisted of a combination of the traditional method of delimiting sites by visual recognition of the material concentration and a systematic geolocation of the characteristic vestiges of the concentrations. The objective was to compare the approximate delimitation which is generally achieved with the measurable area of concentration suggested by the individual location of the vestiges. A good match between the visually estimated areas and those that can be restored through the cloud of points can be discerned (Fig. 2).

Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion Fig. 2 Comparison between the field concentration control (black dotted lines) and the density map generated from Archeotracker data.

A posteriori identification of sites

The individual location of the vestiges present in the topsoil and their spatial representation in the form of density maps can lead to the identification of certain particular concentrations of material which have not been identified in the field (Fig. 3). The identification of these anomalies in the distribution of off-site scatters should normally prompt a return to the field for a verification of these anomalies. However, it should be recalled that the concentration criterion is not the only one to be taken into account in the identification of a site index. The composition (in particular with regard to chronology) of the material which makes up the concentration must also be appreciated, since "artificial" concentrations of material of various origins may well have been generated by post-depositional phenomena (erosion of slopes, accumulation of alluvial material) or be foreign to the history of the actual land use (e.g. resulting from the transport of material from one place to another).

Multiscalar approach: possibility of spatial analysis at the scale of a landscape

The analysis of the occupation dynamics of a rural area relies on the processes underlying the installation and abandonment of settlements as well as on the measurement of the ways of exploiting the surrounding territory. While the modelling of disruption or continuity as a tool of analysis has become a classic, the measurement of the links to unmanaged territory remains a much more complex affair. In France, the considerable advances made in preventive archaeology over the past thirty years have, moreover, provided little progress in this field. Field archaeology remains marginal. However, the possibility of localizing each and every artefact collected on the Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion surface opens the way to a mapping of the densities of off-site material at the intra-plot scale. This approach makes it possible to go beyond the mapping of the density of vestiges generally carried out by parcel (or fragment of a parcel, i.e. the Collecting Units) of a size generally between 2 and 5 ha. It is possible to analyse the distribution of this material with a high spatial resolution. Generally interpreted as the archaeological trace of manure spreading [START_REF] Poirier | Techniques d'amendement agraire et témoins matériels pour une approche archéologique des espaces agraires anciens[END_REF] , this off-site material makes it possible to establish a link between the sites and the landscape by recording the gradient in the intensity of agrarian investment in the landscape over time. A finer interpretation of the distribution and the modalities in variation of this gradient was hitherto limited by the heterogeneity in the size of the collection units. Spatial analyses based on the individual location of the remains in Archeotracker now enable us to generate density maps with a spatial resolution of a few square meters. This is particularly helpful when reviewing the archaeological sites identified -but also of the current habitats -in a wider context of land development. In cases where a high stability of settlement exists, the date of establishment for the current occupation can be inferred by dating the halo of off-site material surrounding it.

The combination of non-invasive methods in the REPERAGE project

The programme described above was developed as part of a project to study the settlement dynamics of the central valley of the Garonne in the south-west of France. This project, entitled REPERAGE, aims to study the processes of human settlement of the river valley and the lower terraces of the Garonne, through the use of, among other methods, systematic prospecting campaigns (Fig. 4).

Following the planned route of a railway track, it aims at developing a comprehensive, noninvasive archaeological strategy which combines high resolution mapping of fieldwalking artefacts, low altitude aerial surveys using unmanned aerial vehicles on-board multispectral and Lidar sensors, and geophysical surveys (GPR, magnetometer). The objective is to obtain a high level of non-invasive knowledge on diachronic settlement systems and dynamics, relationships between past human societies and the natural environment, preceding the implementation of the actual field operations of rescue archaeology. The main historical and archaeological questions of this study are related to the main issues of the medieval period in the South-West of France, and (more specifically) concern the nature of ancient and late-antiquity occupation as well as the establishment of the settlement centres in the Early Middle Ages, the articulation between nucleated and dispersed settlement in the Middle Ages and the medieval organisation of the territory.

Systematic fieldwalking

Since 2014, systematic fieldwalking surveys have covered nearly 650 hectares, most of which lie in the immediate vicinity of the future railway track. These surveys permitted the discovery of about 40 concentrations of surface material which indicate potential archaeological sites dating from Prehistory to the present day (Fig. 5). They have also allowed the collection of nearly 60 000 off-site artefacts, including 15 000 shards of pottery, the only material remnants of manure spreading from Protohistory. The study of the ceramic material is still under way, but the major trends in land use dynamics are in line with the models generally established for France. Sedentary occupation is identifiable from the end of the Bronze Age and during the Iron Age; it expanded and intensified markedly during the Roman period before decreasing slightly during the Middle Ages and the modern period. It must be borne in mind, however, that we have access only to these occupation periods, and that the medieval and modern sites which are still occupied today cannot be taken into account in these calculations. The challenge in our work lies precisely in specifying the establishment dates of these current occupations by observing the halo of off-site scatters which can be detected around them. Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion

Ground Penetrating Radar (GPR)

At the site of Notre-Dame-de-Baude, located in the town of Labastide-du-Temple in our study area, indications that the parish church had existed prior to the founding of the Templars had been identified by F. Hautefeuille in modern written documentation during the 1990s.

It is likely that the building was located on the site of a Gallo-Roman villa, a mosaic of which had been excavated in the 1950s.

A systematic fieldwalking survey of the plot using the Archeotracker application confirmed the presence of a concentration of vestiges from the ancient and medieval periods.

The GPR prospecting of the concentration zone revealed both the ground plan of the ancient buildings and the subsequent establishment of a church on the masonry foundations of the former [START_REF] Gragson | Discovery and appraisal of the early Christian church of Notre Dame de Baudes near Labastide-du-Temple, France[END_REF] .

Magnetic surveys

The use of a GSM-19 magnetometer, generally reserved for the coverage of sites of exceptional interest, was tested on concentrations of surface features, with the aim of specifying the location and nature of any buried vestiges. The two site indices tested, which dated from the Roman period, each revealed the presence of a strong magnetic anomaly in the subsoil testifying to the probable presence of buried structures or buried masses of metal (Fig. 6). On the other hand, we are somewhat disappointed that we have not been able to map more accurately any structures (masonry, ditches). Nevertheless, the use of this methodology at least validates the existence of buried vestiges at the location of the concentrations detected on the surface, and this in a completely non-invasive way.

Drone overflights

In a similar way, we have used the resources of drones carrying different sensors relevant for archaeology during the last four years.

Infrared/Multispectral sensors

First of all, we developed the use of a thermal imager embedded in an octorotor drone (octocopter). On bare soil, the objective is to detect anomalies of the surface temperature of the plots produced by the thermal inertia of the buried remains. Obviously, buried masonry or ditches do not react to changes in temperature at the same rate as the sediment that contains them. On cultivated soil, the infrared reflectance of plants growing above buried vestiges will be different from that of plants growing under normal conditions. The validity of this procedure was first tested on concentrations of Modern Era features identified by pedestrian prospecting whose existence was confirmed by the Napoleonic cadastre [START_REF] Poirier | Low Altitude Thermal Survey by Means of an Automated Unmanned Aerial Vehicle for the Detection of Archaeological Buried Structures[END_REF] . Then, an overview of concentrations of antique and medieval features made it possible to identify infrared reflectance anomalies. The site of Notre-Dame-de-Baude, which we have already mentioned, also allowed the identification of thermal anomalies at the location of the concentration of surface features and the structures identified by Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion the GPR (Fig. 7). Our objective is to increase the number of flights over the same sites, varying the conditions of intervention (soil cover, temperature, humidity), so as to define a reference frame which is as precise as possible regarding the conditions favourable to the identification of Reflectance anomalies. To develop this approach, we equipped ourselves with a fixed wing drone equipped with a multispectral sensor (5 bands) capable of a greater autonomy. The truly multispectral character of the sensor allows us to develop an authentic remote sensing approach similar to that used with satellite images, including the calculation of vegetation indices and the possibility of automating the detection of reflectance anomalies.

Lidar sensor

We also used an opportunity to acquire a multi-rotor drone equipped with a miniaturized Lidar sensor. This equipment makes it possible to document the small forested areas generally not covered by traditional Lidar examinations for reasons of cost (Fig. 8). Indeed, the fixed costs associated with getting the Lidar airborne in an airplane or a helicopter explain why only forested heights of a certain size are affected by Lidar examination. In our case, even if the Lidar sensor is less powerful than a conventional Lidar, lowering the flight altitude to 50 meters can result in a point density equivalent to or better than a conventional Lidar (up to 20 or 30 points per square meter under good conditions). Above all, the flexibility in using the drone makes it possible not only to choose the optimal moment for the overflight but also to repeat overflights over the same sites in order to intensify the coverage of the area. As a result, we were able to document three archaeological sites located in forested parts of our study area. Dating to Antiquity and the Middle Ages, they were known from ancient texts, but the difficulty of accessing them in the dense undergrowth had not previously allowed the drafting of precise plans.

The first case is a site identified at the beginning of the 20 th century as a probable "Roman camp". The Lidar survey now makes it possible to draft a precise plan. It is composed of a trapezoidal enclosure of about 70m to a side, intersected in the south-west by a recent clearing. The accuracy of the measurements makes it possible to determine the width and depth of the ditches. It only remains to collect finds on the surface to specify the dating of this structure, which is again made difficult by the density of the forest cover.

The second case is the site of feudal motte-type castle, also identified at the turn of the 19 th century. While the well-preserved mound (up to 2m in height) had been relatively well described by older authors, the Lidar survey revealed a complex water drainage network to connect this enclosure to the surrounding water system. Sherds of pottery collected on the surface make it possible to date part of this site to the medieval period, and more probably to the Early Middle Ages.

The last example is that of the church of Gavancelle. This place name is mentioned in a list of churches belonging to the diocese of Toulouse at the beginning of the 11 th century. The present place name, "the cross of Gancelle" probably preserves the memory. On the ground, a mound is visible, surmounted by a recent wooden cross. The Lidar coverage of the sector has made it possible to place this mound in its original micro-topographic context. It is possible to measure all the relevant aspects: length, width, and preserved height. Unfortunately, no other clearly interpretable micro-topographic anomaly could be detected around it (neither agricultural vestiges nor remains of masonry). On the other hand, it is clear that the church was located on a plateau which definitely overlooked the area immediately to the east.

Conclusion

Summing up, our approach is that of a non-invasive kind of archaeology aimed at detecting anomalies (spectral or topographical) with the help of drones, validated on the ground by surface collections of artefacts, and supplemented by the mapping of buried remains through geophysics. This archaeology of landscapes and territories will clearly allow us to locate, date, characterize and rank settlements and portions of the landscape used by ancient societies without recourse to the destructive method of excavation.

As regards the reconstruction of the interactions between ancient societies and the Garonne River, there seems to be a variable attitude over time (Fig. 9). In particular, there is a gradual separation of settlement sites from the river from Protohistory to the present day. While the average distance to the Garonne was around 2 km during the protohistoric periods, this increased gradually to reach more than 3 km today. This evolution can also be observed in the average altitude of settlement sites, which tend to rise over time from an average of 82m in Protohistory to an average of 97m during the Middle Ages.

This evolution can only be interpreted in the light of the dynamics which shaped the Garonne river over time [START_REF] Valette | Mise en oeuvre d'une démarche géohistorique pour la connaissance de l'évolution des paysages fluviaux : l'exemple de la moyenne vallée de la Garonne[END_REF] . The resident populations probably sought to protect themselves from the floods Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion and wanderings of the river bed which are clearly attested by the Napoleonic cadastre for more recent times. Indeed, between the end of the 18 th and the end of the 19 th century, the municipalities bordering the river had to redraw the cadastral maps almost every decade due to the shifting of the river banks. We still have a lot of work to do in precisely analysing the long-term evolutions of the settlement pattern. Although the Middle Ages as a whole are fairly well represented, the very beginning of this period and the phase of transition from Antiquity is relatively poorly documented. It is a peculiarity of this southern half of France that it provides only very few sites of the early Middle Ages lying in the open landscape. This peculiarity could be explained either by a longer occupation of Roman settlement sites or by an early and sustained establishment of sites occupied from (Late) Antiquity to this day. The occupation phase of the early Middle Ages would be difficult to discern either way, being obscured by the more obvious vestiges of classical Antiquity or trapped under present-day structures which are difficult to access by excavation or survey. A precise dating of off-site scatters recorded by the Archeotracker application around current structures, and a spatial analysis of the location of ancient and medieval sites should provide some answers to this question.

  Fig. 1 Difference between the number of artefacts recorded via Archeotracker and actual in-field counting.

Fig. 3 A

 3 Fig. 3 A posteriori identification of potential sites.

Fig. 4

 4 Fig. 4 Location of the study area.

Fig. 5

 5 Fig. 5 Map and graph of settlements identified through fieldwalking surveys.

Funde

  Fig. 6 Magnetic survey of two Roman material concentrations.

Fig. 7

 7 Fig. 7 Infrared anomalies.

Fig. 8

 8 Fig. 8 Lidar coverage of forested sites.

Fig. 9

 9 Fig. 9 Distance between sites and the Garonne river / Average altitude of sites.

Funde in der Landschaft -Neue Perspektiven und Ergebnisse archäologischer Prospektion


David Guarin

Sina Marhabaie

Alberto Rosso

Daniel Abergel

Geoffrey Bodenhausen

Konstantin L Ivanov

Dennis Kurzbach

Characterizing Thermal Mixing Dynamic Nuclear Polarization via Cross-Talk between Spin Reservoirs

Dynamic nuclear polarization (DNP) embraces a family of methods to increase signal intensities in nuclear magnetic resonance (NMR) spectroscopy. Despite extensive theoretical work that allows one to distinguish at least five distinct mechanisms, it remains challenging to determine the relative weights of the processes that are responsible for DNP in state-of-the-art experiments operating with stable organic radicals like nitroxides at high magnetic fields and low temperatures. Specifically, determining experimental conditions where DNP involves thermal mixing, which denotes a spontaneous heat exchange between different spin reservoirs, remains challenging. We propose an experimental approach to ascertain the prevalence of the thermal mixing regime by monitoring characteristic signature properties of the time evolution of the hyperpolarization. We find that thermal mixing is the dominant DNP mechanism at high nitroxide radical concentrations, while a mixture of different mechanisms prevails at lower concentrations.

D ynamic nuclear polarization (DNP) is a powerful method for enhancing the polarization of nuclear spins with the aim of boosting weak nuclear magnetic resonance (NMR) signals. In recent years, DNP has experienced a remarkable "renaissance" due to novel methodological developments, in particular for solid-state DNP (MAS-DNP) 1-4 and dissolution DNP (D-DNP), 5-10 which enables many new applications in practically all fields of NMR 10,11 and MRI 5,7 ranging from incell metabolomics to cancer monitoring in humans. At the heart of DNP lies the transfer of electron spin polarization to nuclear spins upon pumping electron paramagnetic resonance (EPR) transitions of stable mono-or biradicals. To widen the range of applications of DNP and to optimize the enhancements, a thorough understanding of the underlying mechanisms is a prerequisite. At this time, five distinct DNP mechanisms are known to play a role in solids: 12-14 the solid effect (SE), the differential solid effect (DSE), the cross-effect (CE), thermal mixing (TM) and the Overhauser effect (OE). While the latter exploits dissipative cross-relaxation, the former four mechanisms make use of coherent polarization transfer. The SE is due to pumping "forbidden" combinations of EPR and NMR transitions; since it involves an isolated electron spin and an isolated nuclear spin, this mechanism is most efficient when 50 using dilute monoradicals. A more efficient DNP mechanism 51 that dominates for dilute biradicals or concentrated mono-52 radicals is provided by the CE, which requires a pair of electron 53 spins so that "triple spin flips" (of two electron spins, SS′, and 54 one nuclear spin) can occur (denoted SS′I for I = 1 H, or SS′I′ 55 for other nuclei I′ such as 2 H, 13 C, 31 P, etc.) Finally, 56 concentrated monoradicals with strong intermolecular inter-57 actions can lead to TM, a mechanism for which thermodynamic 58 concepts can be used 15-17 to describe polarization transfer in 59 terms of heat exchange between different reservoirs. TM is 60 effective only when the EPR spectral width Δν e , given either by 61 homogeneous line broadening or electronic spectral diffusion 62 (eSD), exceeds the nuclear Zeeman interaction frequency 63 ν I . 18,19 64

Various definitions for TM have been given. [14][15][16][17] Here we 65 consider that TM entails three important properties. (1) 66 Electron spins, even under microwave irradiation, display a 67 behavior that can be described using thermodynamic concepts. 68 Namely, their description involves two heat reservoirs: a

69 Zeeman reservoir, introduced to describe energy variations on 70 the order the electronic Zeeman interaction ν e , and a non-71 Zeeman or dipolar reservoir, to describe exchange of energy on 72 the order of the relevant width Δν e of the EPR spectrum. (2) 73 Triple-spin flips SS′I can establish a contact between the 74 nuclear Zeeman and electron non-Zeeman reservoirs, 19 since 75 they involve the transfer of energy of the order of the nuclear 76 Zeeman energy ν I between nuclear and electron spins. (3) The 77 rates of triple-spin flips are fast compared to the rates of other 78 phenomena such as electron or nuclear relaxation rates.

79

Only if these properties are fulfilled can DNP proceed 80 predominantly via TM. In this case, the hyperpolarization 81 process displays two characteristic features: In practice, DNP often results not from a single dominant 98 mechanism, but from a combination of different processes such 99 that it may be difficult to separate contributions of different 100 mechanisms. 16,[20][21][22] A common strategy for the analysis of 101 DNP processes is to simulate experimental results and to assess 102 the underlying mechanism by comparing simulations and 103 experiments. However, this approach is notoriously difficult 104 even for discriminating SE and CE contributions. 19 it to the case of amorphous solids that are used in modern DNP applications. 18 In this experiment, we polarized two different nuclei (here I = 1 H and I′ = 2 H, [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C or 31 P), stopped saturating the EPR transitions, then depolarized one of the nuclei by rf saturation, and observed the subsequent time-dependence of polarizations P(I) and P(I′). This includes a possible spontaneous transfer of polarization between nuclear spin species via the dipolar electron reservoir, which is a clear indication of so-called triple spin flips (SS′I and SS′I′ flips).

These two experiments hence allowed us to monitor to determine whether TM was the dominant mechanism for DNP or not. 25:25:25:25% in volume). Sample 4 comprised the same 147 solvent mixture of sample 1, but contained 0.5 M K 2 HPO 4 in 148 addition. We used the nitroxide TEMPOL as characterized by a 149 broad inhomogeneous EPR line (ca. 0.5 GHz). The TEMPOL 150 concentration was varied in the range of 10-100 mM. 151 DNP. Our DNP apparatus is described in detail in 152 references. [24][25][26] All samples were immersed in liquid helium 153 at 4.2 K in the cryostat at atmospheric pressure. An ELVA1 154 microwave source coupled to a Virginia Diodes (VDI) 155 frequency doubler provided microwaves (μw) at a frequency 156 ν μw = 187.9 GHz for all experiments A and B. The μw-field was 157 modulated over a range of 100 MHz using a sawtooth 158 modulation function with a 1 kHz repetition rate. The 159 microwave frequency profiles were obtained by stepping the 160 central frequency over a range 187.8 < ν μw < 188.4 GHz with a 161 20 MHz step size and the same modulation bandwidth.

162

We used two-channel NMR probes to detect the polarization 163 of both protons and heteronuclei I′ = 2 H, [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C or 31 P at B 0 = 6.7 164 T. Although TM-DNP leads to higher nuclear polarizations 165 P(I) at 1.2 K, the sample temperature was set to 4.2 K to speed 166 up the polarization build-up and shorten nuclear spin relaxation 167 times, so that the experiments could be run faster. Thus, the 168 inverse sample temperature (and electron spin temperature 169 before microwave saturation) at thermal equilibrium was 1/T 0 170 = 0.25 K -1 .

171

In experiment A, the nuclei I = 1 H and I′ = 13 C were 172 polarized through continuous-wave (cw) μw irradiation to 173 determine the steady-state polarization and the characteristic 174 build-up times of the nuclei. After achieving the steady state, 175 the μw irradiation was interrupted to determine T 1 (I) and 176 T 1 (I′) decay times. Experiment B consisted of polarizing 1 H 177 nuclei to their steady state by cw μw irradiation prior to 178 switching off the μw source. The 13 C nuclei were then 179 selectively saturated and the time evolution of both 1 H and [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C 180 nuclei was subsequently monitored in the absence of μw-181 irradiation. Saturation was achieved by a series of NMR pulses.

182

In all experiments, 1°detection pulses were used for I = 1 H 183 and 5°pulses for I′ nuclei at intervals of 5 s to monitor the 184 signal intensity as a function of time.

■ RESULTS AND DISCUSSION

186 To elucidate the limits of TM, we systematically varied the 187 experimental conditions for experiments A and B, to probe the 188 two characteristic signatures (i) and (ii) listed above.

β I = 1/k B T I , is ω β ω = - ℏ = - ℏ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ P I k T ( ) tanh 2 tanh 2 I I I I B ( 1
)
where ω I is the nuclear Zeeman resonance frequency in the external field. It is readily seen in Figure 1b that at TEMPOL concentrations above 50 mM, both nuclei converge to a common spin temperature after the initial build-up. In particular, 1/k B T I ( 13 C) reaches a maximum when the TEMPOL concentration is 50 mM. Most importantly, at lower radical concentrations (10-40 mM), the [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C spin temperature is considerably higher than the proton one, thus indicating that TM is not the dominant mechanism since condition (i) listed above is not fulfilled anymore. [START_REF] Abragam | Principles of Dynamic Nuclear-Polarization[END_REF] The fact that TM no longer dominates at low radical concentrations originates from reduced rates of electronelectron flip-flops as the distance between radical centers becomes larger. On the one hand, when electron spectral diffusion slows down due to decreasing flip-flop probabilities, the electron spins cannot be treated anymore using thermodynamic concepts. Indeed, in this limit, the microwave To our surprise, the transfer of polarization between the two 264 nuclear reservoirs is not entirely suppressed, even at a radical 265 concentration as low as 10 mM. Thus, even though the 266 signature features of a common spin temperature and an 267 efficient heat transfer are not fulfilled, we observe triple spin 268 flips, indicating a non-TM regime with a reduced rate of such 269 transitions.

Through this, at radical concentrations of 25 mM and below we observe an "anomalous" heat transfer as the maximum inverse spin temperature 1/k B T I (C13) is reached at a time where 1/k B T I ( 1 H) is still higher. These data clearly confirm the presence of triple spin flips and transfer of polarization between the Zeeman reservoirs of the 1 H and [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C nuclei and the non-Zeeman electronic reservoir, but point to the fact that the rates of triple spin flip transitions involving [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C spins are comparable to or lower than their nuclear relaxation rates, impeding the prevalence of TM as dominant DNP mechanism.

In conclusion, at TEMPOL concentrations of 50 mM or above all characteristic features of TM, as defined above in points (i) and (ii), are observed, while at lower concentrations the nuclear reservoirs do not reach a common spin temperature; the heat transfer is inefficient despite the occurrence of triple spin flips.

The characteristic build-up times and steady-state spin temperatures at the end of the build-up (experiment A) as well as the transfer times (characteristic cooling times of the depend the properties of the EPR spectrum of TEMPOL. [START_REF] Abragam | Principles of Dynamic Nuclear-Polarization[END_REF]19 However, it is readily seen that the profiles for 1 H and 2 H are similar, while they differ significantly from those of 13 C and 31 P, which show slight differences between each other. In cases of CE or SE, one would expect a frequency difference of ν I or 2ν I , respectively, between the maxima and minima of the microwave profiles and thus a strong dependence on the nuclear gyromagnetic ratio.

Since none of these features is observed for any nuclei, we assume a mixture of different mechanisms for the low radical concentration regime. For 1 H and 2 H we observe profiles where the extrema are separated by ca. 440 MHz (more than the 315 proton Larmor frequency, which is ν I = 285.3 MHz at 6.7 T), 316 and for [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C and 31 P the extrema are separated by ca. 300 MHz. 317 Vega, Han, and co-workers [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF]14,16 have shown, based on 318 simulations of experimental data under similar conditions, 319 that at 25 mM TEMPOL for 1 H, a combination of CE and DSE 320 can reproduce the experimental observations, in agreement 321 with our observation that the 1 H and 13 C spin reservoirs do not 322 reach a common spin temperature at this radical concentration. 323 This indicates that the description of the DNP process as 324 "pure" TM breaks down. We observe a similar behavior for 2 H, 325 13 C and 31 P. Note that the difference between the μw profiles 326 characteristic for TM and CE is very subtle (the μw profiles for H can be reproduced both with TM-based as well as CE-based 328 models). 13,24 329 Interestingly, we observe for all these nuclei triple spin flips f5 330 via experiment B at 25 mM TEMPOL (see Figure 5).

331

For pairs of I = 1 H with I′ = 13 C, 2 H or 31 P, one finds a 332 spontaneous heat transfer via the e NZ reservoir (see Figure 5a,b 333 for the spin temperature as a function of time for I′ = 2 H and 334 31 P). This clearly shows that the presence of triple spin flips is 335 not a sufficient condition for TM, though a necessary one, if we 336 assume TM as strictly defined by the signature features listed 337 above. 338 Additionally, in experiment B, the heat transfer between [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C 339 and 1 H via the e NZ reservoir with 50 mM TEMPOL works both 340 ways (see Figure 5c), which confirms the consistency of our 341 interpretation. In contrast, radicals with narrow EPR lines like 342 50 mM BDPA do not lead to any observable heat transfer in 343 experiment B. This excludes direct cross-talk between the 344 nuclear reservoirs (data not shown). indicates the breakdown of SS′I transitions), the DSE is the most likely mechanism, as pointed out by Han and coworkers. [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] From theoretical considerations, we expect the TM regime to establish at even lower radical concentrations if the sample temperature or the external magnetic field are decreased.

Considering that processes, which involve a heat flow, need to compete with electronic T 1 relaxation, it appears intuitive that conditions that prolong relaxation favor thermal mixing.

From the experimental point of view, it is important to note that the lowest [START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C spin temperature can be reached at TEMPOL concentrations of 50 mM due to low 1 H spin temperatures and the dominance of TM. Yet, even lower 1 H temperatures can be reached at a lower TEMPOL concentration of 40 mM because favorable saturation factors. 29,30 This information is crucial for users who cannot rely on crosspolarization techniques. 26 In this situation TM-DNP can be a remedy, since it not only allows one to hyperpolarize protons, but various other nuclei by exploiting additional pathways for efficient polarization transfer.

■ AUTHOR INFORMATION

Corresponding Authors *E-mail: kurzbach@ens.fr (D.K.).

  82 (i) the spin temperatures T I of all nuclear spin species 83 converge to a single common value for long times t → ∞ 84 in the presence and absence of microwave irradiation. As 85 a result, all nuclei I for which ν I < Δν e are "cooled down" 86 in the same way at all microwave frequencies, in contrast 87 to SE and CE where the microwave frequency for most 88 efficient DNP depends on the nuclear gyromagnetic 89 ratio, γ I . 90 (ii) A second important consequence of TM is that, if 91 different types of nuclei are present in the sample, heat 92 can flow spontaneously from one nuclear reservoir to 93 another via the non-Zeeman electron reservoir. In other 94 words, if TM is predominant, polarization transfers 95 between different nuclear species are expected to follow 96 the simple laws of heat propagation.97

  When TM comes into play, the superposition of various mechanisms becomes even more difficult to unravel. Therefore, it is currently not clear what DNP mechanisms are active for the frequently used nitroxide-radicals, 23 in particular at high magnetic fields B 0 > 6 T at T < 1.2 K for D-DNP or 20 T at 100 K for MAS-DNP. Here we propose an experimental approach that allows one to determine whether TM is the predominant mechanism in state-of-the-art systems employing nitroxide radicals, based on probing the above-mentioned characteristic features (i) and (ii). To elucidate the active DNP mechanism and to quantify the flow of polarization between different heat reservoirs via triple spin flips, we performed DNP experiments on spin systems comprising protons I = 1 H and other nuclei (I′ = 2 H, 13 C and 31 P) in the presence of radicals at variable concentrations. The experiments were carried out in two different ways: (A) we monitored the build-up curves of the polarization P(I) and P(I′) of two different nuclei I and I′ in the presence of microwave saturation of the EPR spectrum, and subsequently measured their return to thermal equilibrium after interrupting the microwave irradiation; (B) we performed in a systematic way an experiment proposed by Goldman and coauthors for single crystals of LiF (where I = 7 Li and I′ = 19 F), and adapted

Figure 1 .

 1 Figure 1. (a) Energy reservoirs involved in experiment A that monitors the buildup of the polarizations P(I = 1 H) and P(I′ = 13 C) after switching on the saturation of the EPR spectrum by microwaves (μw), and their decay after interruption of the μw irradiation. The μw irradiation cools down the non-Zeeman dipolar electron reservoir e NZ ; as a result, the 1 H and 13 C spins are polarized simultaneously via heat exchange between the reservoirs. Nuclear relaxation proceeds via dissipation of energy to the lattice. (b) DNP build-up curves and decay of polarizations after switching off the μwfield (at the delays indicated exemplarily for 1 H and 13 C in the panel of 100 mM TEMPOL) for a sample containing I = 1 H and I' = 13 C nuclei. The polarizations are expressed in terms of inverse spin temperatures, 1/T I . Traces shown in lighter color indicate the ranges of experimental errors.

f1 189 ( 1 )

 1891 Reaching a Common Spin Temperature. Figure 1a displays 190 schematically the flow of spin order in experiment A. Our μw 191 polarizes the non-Zeeman dipolar electronic (e NZ ) energy levels 192 by cooling down the corresponding heat reservoir. Con-193 sequently, triple spin flips tend to equalize the e NZ spin 194 temperature and the nuclear spin temperature of both 1 H (SS′I 195 flips) and 13 C (SS′I′ flips) heat reservoirs. After switching off 196 the μw irradiation, both nuclear polarizations P( 1 H) and P( 13 C) 197 decay and equilibrate at the lattice temperature. Typical DNP 198 build-up curves of these nuclear spin polarization and 199 subsequent decays, obtained for different concentrations of 200 TEMPOL, are shown in Figure 1b (sample 2). At all radical concentrations, the nuclei I = 1 H and I′ = 13 C have different DNP buildup times and different relaxation times. The polarizations are expressed in units of 1/T I , the inverse spin temperature of the corresponding nucleus. The relation between the nuclear spin polarization, P(I), and the inverse spin temperature

( 2 )

 2 Figure 2a. Triple spin flips offer a mechanism through which the transfer of polarization, i.e., of heat from one nuclear reservoir to another via the e NZ reservoir, can take place. Hence, a spontaneous heat flow from a warmer to a colder spin reservoir attests for the presence SS'I triple-spin flips. Through our experimental setup, we could observe the transfer of polarization from 1 H to 13 C, i.e., as the 1 H reservoir heats up, the[START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C reservoir cools down. When the rate of triple spin flips is high, the two nuclear and the e NZ reservoirs reach a common spin temperature before they relax to the equilibrium temperature. This is exactly what is observed in Figure2bbetween 40 and 100 mM, where the 13 C polarization of sample 2 reaches a maximum when the temperatures of the two nuclear reservoirs become equal (the time traces cross). As such a polarization transfer proceeds via the electronic non-Zeeman reservoir, it can be investigated experimentally by stepping away from ideal TM conditions, e.g., by using lower

f3 13 C

 13 reservoir in experiment B) are summarized in Figure 3. Note that heat transfer becomes faster with increasing radical concentration (Figure 3c), thereby confirming the dependence of the triple spin flip rate on the TEMPOL concentration. Changing Hyperpolarized Nuclei. To shed more light on the mechanistic details at intermediate TEMPOL concentrations, where features (i) (similar spin temperatures) and (ii) (characteristic heat transfer) of TM are not fulfilled, but triple spin flips are still observed via experiment B, we measured the dependence of the spin polarization P(I′) of the four nuclei I′ = 1 H, 13 C, 2 H and 31 P on the microwave irradiation frequency ν μw f4 at 25 mM TEMPOL. The profiles are shown in Figure 4. When TM is dominant, the widths of the μw-profiles would primarily

Figure 2 .

 2 Figure 2. (a) Energy reservoirs involved in experiment B that monitors the transfer between the polarizations P(I = 1 H) and P(I′ = 13 C) after the interruption of the μw irradiation and saturation of either I or I′ nuclear reservoirs. Via triple spins flips, the polarization flows from 1 H to the non-Zeeman dipolar electron reservoir, and is then transferred to 13 C spins via SS′I′ triple spin flips. These processes are accompanied by relaxation to the lattice (omitted in the flow diagram). (b) Time evolution of the inverse nuclear spin temperature 1/T I . First, a polarization P(I = 1 H) is generated by DNP.Subsequently, after the μw irradiation is interrupted, the[START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C spins are saturated, and a spontaneous flow of polarization from 1 H to[START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C reservoirs is observed. Spontaneous transfer of heat from the 1 H to[START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C reservoir is indicative of triple spin flips SS′I and SS′I′.

Figure 3 .

 3 Figure 3. (a) Characteristic build-up times, t BU for P( 1 H) and P( 13 C) polarizations at different TEMPOL concentrations, obtained by fitting experimental build-up curves to monoexponential functions. At low radical concentrations, the buildup of P( 13 C) is faster than the buildup of P( 1 H), indicating a change of the dominant DNP regime (see text for details). (b) Dependence of the inverse steady-state spin temperatures 1/T I ( 1 H) and 1/T I ( 13 C) as a function of the radical concentration. At a TEMPOL concentration of 50 mM, these inverse spin temperatures are similar within experimental error, indicating that TM is predominant, while below 40 mM this is no longer the case. (c) Characteristic polarization transfer times from P( 1 H) to P( 13 C) obtained by fitting the first part of the 13 C time dependence in experiment B to monoexponential build-up functions. The dashed lines are to guide the eye.

■

  CONCLUSIONS Combining experiments A and B yields a strategy for the determination of TM contributions to DNP, corresponding to the limiting case where the spin temperatures of all spin reservoirs tend to a common value T I for t → ∞ due to triple spin flips and the spontaneous heat transfer between different reservoirs. This strategy allows one to assess the presence of the TM regime, understood as a situation where a common nuclear spin temperature emerges from a spontaneous heat flow, through triple spin flip transitions. These two necessary conditions must both be fulfilled to establish TM, while one of them alone is insufficient. We want to stress that the sole occurrence of triple spin flips, or of spontaneous heat transfer, is not indicative of strict TM, if the spin temperatures of the different nuclei in the system do not converge. It can be clearly stated that TM is the dominant mechanism at high TEMPOL concentrations (≥50 mM) at 4.2 K and 6.7 T, i.e., for experimental conditions frequently used in modern DNP experiments, while for lower TEMPOL concentrations (≤40 mM) the spin temperatures of the different reservoirs can be different. The latter fact, in combination with the observation of spontaneous heat transfer, indicates the presence of SS′I flips outside of the TM regime, which corresponds to a mixture of different mechanisms. At TEMPOL concentrations of 10 mM, where we observe only very weak heat transfer (experiment B

Figure 4 .

 4 Figure 4. Dependence of nuclear signal intensities on the microwave frequency ("μw profiles") detected for 1 H (sample 1), on 13 C (sample 2), on 2 H (sample 3) and on 31 P (sample 4). In all cases, the frequency differences between the maxima and minima of the μw profiles do not correspond to multiples of ω I , indicating significant contributions of TM; however, slight differences between the profiles indicate admixtures of SE and CE.

Figure 5 .

 5 Figure 5. Experiments B for (a) sample 2 and (b) sample 4.For both 2 H and 31 P a spontaneous polarization transfer from 1 H can be observed with 25 mM TEMPOL. (c)[START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C to 1 H polarization transfer in experiment B for sample 2 containing 50 mM TEMPOL after polarizing[START_REF] Leavesley | Effect of electron spectral diffusion 458 on static dynamic nuclear polarization at 7 T[END_REF] C and saturating 1 H. Evidently, triple spin flips can transfer heat in both directions.

142

  Sample Preparation. Sample 1 consisted of glycerol-d 8 , D 2 O and 143 H 2 O (50:40:10% in volume). Sample 2 consisted of the same 144 mixture, with additional 1.5 M pyruvate-1-13 C. Sample 3 145 consisted of glycerol, glycerol-d 8 , D 2 O, and H 2 O (with ratios 146

*E-mail: ivanov@tomo.nsc.ru (K.L.I.). ORCID Dennis Kurzbach: 0000-0001-6455-2136

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors thank Dr. Maurice Goldman for helpful discussions and Bruker BioSpin for providing a prototype of the DNP equipment. This work was supported by the French CNRS and the European Research Council contract "dilute para-water". K.L.I. acknowledges ENS (Paris) for a guest professorship and the Russian Foundation for Basic Research (Grant No. 17-03-00932). A.R. acknowledges ANR-16-CE30-0023-01 (THERMOLOC).


Angelo Troia
email: angelotroia@unipa.it

Germinal Rouhan

A Braun

I Boryana Durieu

I Delalandei

J Lloyd

Olympica

Clarifying the nomenclature of some Euro-Mediterranean quillworts (Isoetes, Isoetaceae): indicator species and species of conservation concern

Keywords: conservation, Europe, Isoetes, lycophytes, Mediterranean, taxonomy

Clarifying the nomenclature of some Euro-Mediterranean quillworts ( Isoetes , Isoetaceae): Indicator species and species of conservation concern Angelo Troia, Germinal Rouhan

INTRODUCTION

The lycophyte genus Isoetes L. includes approximately 250 extant species distributed worldwide [START_REF] Troia | The genus Isoetes (Isoetaceae): a provisional checklist of the accepted and unresolved taxa[END_REF][START_REF] Ppg | A community-derived classification for extant lycophytes and ferns[END_REF] that are the remnants of one of the most ancient extant lineages of vascular plants, diverged from Selaginella P.Beauv. as early as the Devonian Era [START_REF] Pigg | Isoetalean lycopsid evolution: from the Devonian to the present[END_REF][START_REF] Larsén | Disentangling the phylogeny of Isoetes (Isoetales), using nuclear and plastid data[END_REF]Klaus & al., 2017). For this reason, they are of great evolutionary importance [START_REF] Karol | Complete plastome sequences of Equisetum arvense and Isoetes flaccida: implications for phylogeny and plastid genome evolution of early land plant lineages[END_REF][START_REF] Doyle | Phylogenetic analyses and morphological innovations in land plants[END_REF][START_REF] Petersen | Why did heterospory evolve?[END_REF]. They also have significant ecological importance because their presence in aquatic habitats such as lakes, rivers and temporary wetlands serves as indicators of superior ecological conditions in those habitats (e.g. Bagella & Caria, 2013;[START_REF] Lumbreras | Assessing the conservation status of Mediterranean temporary ponds using biodiversity: a new tool for practitioners[END_REF][START_REF] Sciandrello | Diversity and spatial patterns of plant communities in volcanic temporary ponds of Sicily (Italy)[END_REF]. These species are also of conservation concern given that most are rare or threatened; for example, García [START_REF] García Criado | European Red List of Lycopods and Ferns[END_REF] report that 10 out of 20 species occurring in Europe have been assessed as threatened.

In the western Mediterranean area, Isoetes longissima Bory (= I. velata A.Braun, following [START_REF] Troia | A critical conspectus of Italian Isoëtes (Isoetaceae)[END_REF] is one of the physically largest and most significant Isoetes species, both phylogenetically (it is nested in a clade including not only Mediterranean, but also African, Indian and Western North American taxa; [START_REF] Larsén | Disentangling the phylogeny of Isoetes (Isoetales), using nuclear and plastid data[END_REF][START_REF] Pereira | Origin and biogeography of the ancient genus Isoëtes with focus on the Neotropics[END_REF] and ecologically (e.g. [START_REF] Grillas | Mediterranean Temporary Pools; Volume 1 -Issues relating to conservation, functioning and management[END_REF]. It is recognized, however, to be 'a taxonomically difficult suite of species, which merits further research' (Christenhusz & al., 2017). For the purposes of this study, hereafter we refer to this species as I. longissima s.l. or the I. longissima group.

Isoetes longissima s.l. includes useful indicator species for Mediterranean seasonal wetland ecosystems, or 'Mediterranean temporary ponds' that are a conservation priority habitat according to the European 'Habitats' Directive (European Commission, 2013). Mediterranean temporary ponds (and, in general, 'temporary waters', following [START_REF] Williams | The biology of temporary waters[END_REF] are of major conservation concern because, despite their small size, they shelter many rare and endangered species of both flora and fauna [START_REF] Grillas | Mediterranean Temporary Pools; Volume 1 -Issues relating to conservation, functioning and management[END_REF]. They are experiencing an alarming rate of decline and population degradation [START_REF] Zacharias | Mediterranean temporary ponds. A disappearing ecosystem[END_REF].

Due to their small size and simple community structure, temporary pools are often considered as early warning systems of the ecological implications of long-term changes in larger aquatic systems [START_REF] De Meester | Ponds and pools as model systems in conservation biology, ecology and evolutionary biology[END_REF]. Thus, species of Isoetes are good indicators for endangered habitats, but they themselves are species of conservation concern. Isoetes boryana Durieu, for example, is listed in Appendix 1 of the Convention on the Conservation of European Wildlife and Natural Habitats ('Bern Convention') and in the annexes II and IV of the mentioned 'Habitats' Directive. It and the other European Isoetes tenuissima have been assessed as 'Endangered' according to IUCN criteria (Christenhusz & al., 2017, as well as Rouhan & Christenhusz, 2017).

The Isoetes histrix Bory group is another taxonomically complicated group (Bagella & al. 2015[START_REF] Troia | A critical conspectus of Italian Isoëtes (Isoetaceae)[END_REF], 2015a). In addition to I. histrix, it includes several taxa whose taxonomic rank and morphological distinctness need further investigations, all occurring in seasonally waterlogged soils.

As a general rule, species of Isoetes are very difficult to distinguish by general morphological appearance because of their simple, conserved morphology, providing few usefully distinguishing characters. Attempts were made in the past to arrange Isoetes species in groups based on ecological or morphological criteria: for instance, the Braun's system used habitats to distinguish aquatic, amphibian and terrestrial species (Grenier & Godron, 1855), while the Pfeiffer's 'modern' system is based on the ornamentation of the megaspores [START_REF] Pfeiffer | Monograph of the Isoetaceae[END_REF]. Recent phylogenetic studies (e.g. [START_REF] Larsén | Disentangling the phylogeny of Isoetes (Isoetales), using nuclear and plastid data[END_REF] show how artificial these systems are and that they are unable to correctly reflect phylogenetic relationships because of the previously mentioned conserved morphology. Similarly, I. andicola (Amstutz) L.D. Gómez was originally treated in the distinct genus Stylites Amstutz on the grounds of its morphology, but was later moved into Isoetes when both anatomical [START_REF] En Gómez-Pignataro | Vegetative reproduction in a Central American Isoetes (Isoetaceae). Its morphological, systematic and taxonomical significance[END_REF] and molecular evidence [START_REF] Larsén | Disentangling the phylogeny of Isoetes (Isoetales), using nuclear and plastid data[END_REF] showed that it was deeply nested within the large American clade. An accurate study of morphology, ideally considering other available data from anatomy, ecology, genetics etc., is therefore the basis for any taxonomic analysis.

In this study, we examined taxa within the two groups mentioned above, I. longissima (s.l.) and I. histrix (s.l.), with special reference to those taxa (described from France, Algeria and Turkey) that are in need of a taxonomic revision. Taxa from Spain, Italy and Greece have been studied by several authors in recent years [START_REF] Prada | El genero Isoetes L. en la Peninsula Iberica[END_REF][START_REF] Romero | Isoetes fluitans sp. nov.: The identity of Spanish plants of 'I. longissimum[END_REF][START_REF] Romero | A morphometric study of three closely related taxa in the European Isoetes velata complex[END_REF][START_REF] Troia | A critical conspectus of Italian Isoëtes (Isoetaceae)[END_REF]Troia & Greuter, 2015b), and all the other names in those groups have already been typified.

The taxonomic status of Isoetes boryana, a species of great conservation concern, is especially unclear. It is uncertain if it is a 'good species' (as reported in the 'Habitats' Directive, and by other sources such as [START_REF] Christenhusz | Lycopodiophytina. In: Euro+Med Plantbasethe information resource for Euro-Mediterranean plant diversity[END_REF] or a synonym of other species (as suggested by e.g. [START_REF] Prelli | Les Fougères et plantes alliées de France et d'Europe occidentale[END_REF]Romero &[START_REF] Romero | A morphometric study of three closely related taxa in the European Isoetes velata complex[END_REF].

As a first step, we studied dried specimens preserved in several European herbaria, with the primary aim of stabilizing the application of names by typifying them. Thanks to scanning electron microscopy (SEM) investigations of spores removed from the types, it was possible to draw realistic morphological and taxonomic conclusions from original material or from specimens collected in the loci classici (topotypes)

MATERIALS & METHODS

Our study is based on relevant literature and on the herbarium collections, especially those housed in the Paris Herbarium (P), with significant additional data deriving from B, FI and PAL, and from selected specimens in ANG, BM, G, GOET, KFTA, MO, MPU, NTM, US, W (acronyms according to [START_REF] Thiers | Index herbariorum: a global directory of public herbaria and associated stai[END_REF].

For some critical taxa, megaspore and (whenever possible) microspore features were observed with the assistance of SEM imagery. Spores were transferred with dissecting needles from herbarium specimens to aluminium SEM stubs coated with an asphalt adhesive. The stubs were then coated with gold/palladium in a sputter-coater for 2.5 min, and spores were examined using a JEOL 840 A SEM microscope, equipped with an image-digitising system (SEMAFORE software) at the Muséum national d'Histoire naturelle (MNHN) in Paris, France. The accelerating voltage was 10.0 kV. Some other samples were observed and photographed at the University of Palermo, as above, using an Oxford Leo 440 SEM. Terms used for describing the ornamentation of megaspore and microspore perines follow [START_REF] Lellinger | A classification of spore ornamentation in the Pteridophyta[END_REF].

We employed the following species concept: a morphologically and/or ecologically distinct population (or populations), represented by all the specimens available in collections (even if few), is tentatively treated as a distinct species (considering these traits as evidence of the 'existence as a separately evolving metapopulation lineage ', according to De Queiroz, 2007). Clarification of these designations awaits additional data from other sources and in particular data from further observations of living plant populations, and from the addition of molecular phylogenetic evidence. In this sense, we are essentially following the 'Typological Species Concept', sometimes with sufficient confidence to satisfy a 'Morphological Species Concept' while aiming for representation of a more complete 'Biological Species Concept' [START_REF] Hickey | The species concept in Pteridophyta with special reference to Isoetes[END_REF].

Because of paucity of morphological characters for distinguishing species within the genus, we found it difficult if not impossible to assess (phylo)genetic affinities to a sufficiently fine enough level to refer one taxon as a subspecies of another. Such limitations likely explain why, with few exceptions (e.g. [START_REF] Brunton | Isoetes melanopoda spp. silvatica (subsp. nov.), a new quillwort (Isoetaceae) from eastern North America[END_REF], infraspecific ranks so frequently employed in the past (e.g. [START_REF] Engelmann | The Genus Isoetes in North America[END_REF][START_REF] Pfeiffer | Monograph of the Isoetaceae[END_REF][START_REF] Proctor | Isoetes riparia and its variants[END_REF] have recently been used scarcely in Isoetes (e.g. [START_REF] Christenhusz | Lycopodiophytina. In: Euro+Med Plantbasethe information resource for Euro-Mediterranean plant diversity[END_REF].

RESULTS

Results are arranged according to the chronological order of publication of names, partitioned into two taxonomic groups: the Isoetes longissima group and the I. histrix group. For each name, we report information about types (designating lectotypes and epitypes whenever appropriate) and assess taxonomic value. SEM spore images are presented and commented upon whenever appropriate. Raus, 2014;[START_REF] Troia | The genus Isoetes (Isoetaceae): a provisional checklist of the accepted and unresolved taxa[END_REF]. The main distinguishing character shared by all of these taxa is the megaspore ornamentation. From an ecological point of view, the group includes both amphibian and aquatic species. A few taxa hitherto studied in molecular phylogenetics show close evolutionary relationships [START_REF] Larsén | Disentangling the phylogeny of Isoetes (Isoetales), using nuclear and plastid data[END_REF][START_REF] Pereira | Origin and biogeography of the ancient genus Isoëtes with focus on the Neotropics[END_REF], but most of the taxa here listed have still to be included in such analyses.

Isoetes longissima group

Most species that have been studied karyologically (I. asturicensis, I. longissima subsp. longissima, I. olympica) are diploid, with the only exception being I. fluitans, which is a tetraploid [START_REF] Troia | The genus Isoetes (Isoetaceae): a provisional checklist of the accepted and unresolved taxa[END_REF]. Apart from the taxa here investigated, all other names listed above have already been typified elsewhere (in the respective protologues for the recently described taxa, or in [START_REF] Troia | A critical conspectus of Italian Isoëtes (Isoetaceae)[END_REF]. Oct. 1844 by Durieu) indicating that Bory did not see this material until after the name was published. Another specimen collected by Durieu on 12 June 1844 (P00214778), cannot be categorically ruled out as original material, but it is unlikely given that the specimen was collected only a few days before the publication of the name. Specimens MPU015448, MPU015449, MPU015450 (and probably many other specimens in several herbaria) were collected by Durieu on 9 June 1841. These are compatible with the publication date of the new variety, but are labelled 'Isoetes adspersa', a synonym for Isoetes longissima that was used in the 'Flora selecta exsiccata' by Magnier. Accordingly, there is no evidence that Bory saw and used those specimens for the description validating I. setacea var. peyrremondii. We refrain from neotypifying this latter name because we cannot exclude the possibility that undetected original material still exists.

Isoetes longissima

A few years after the description of Isoetes setacea var. peyrremondii, Bory raised it to the species level (as I. capillacea Bory, see below).

= Isoetes capillacea Bory, in Compt. Rend. Hebd. Séances Acad. Sci. 23: 620 (619-).

1846 -Type: not designated.

Bory decided that his Isoetes setacea var. peyrremondii was worthy of a species rank, basing this new species on the previously described variety and on its type (International Code of Nomenclature (ICN) art. 7.4, [START_REF] Mcneill | International Code of Nomenclature for algae, fungi and plants (Melbourne Code) adopted by the Eighteenth International Botanical Congress Melbourne, Australia[END_REF].

Unfortunately, as discussed above, we did not find unambiguous original material.

Our review of Bory's (1844) diagnosis, in conjunction with our study of herbarium specimens identified by him and representing this taxon, provide us with no evidence of a clear morphological distinction between this taxon and Isoetes longissima (s. str.). On that basis, we do not recognize this as distinct from I. longissima (this latter name having nomenclatural priority). According to ICN art. 38.8 (McNeill & al., 2012), the name of the species is validly published because it was accompanied by an illustration 'with analysis', i.e. with details aiding identification. According to ICN art. 9.8 [START_REF] Mcneill | International Code of Nomenclature for algae, fungi and plants (Melbourne Code) adopted by the Eighteenth International Botanical Congress Melbourne, Australia[END_REF], however, an epitype has been designated to serve as an interpretative type, because many morphological and microscopic details cannot be verified on the illustration chosen as lectotype.

According to the details shown in the illustration that is part of the original material and here selected as lectotype, the following diagnostic traits should separate Isoetes adspersa from I. longissima: velum reduced (vs. almost complete); microspores non aculeate (vs. aculeate); sporangia spotted (vs. non spotted); macrospores smaller. In fact, our observations of the remaining original material suggest that macrospore and microspore distinctiveness cannot be distinguished (neither in size nor in ornamentation) (Fig. 1). The velum in the two taxa is variable in extent but always present. The presence of spotted sporangia seems to be the only consistently distinctive feature, but we occasionally observed such pigmentation in other taxa also, such as I. boryana, I. tenuissima, I. tiguliana. Finally, the ecology of the two taxa is identical.

Further work is needed on both fresh and dried material (epitype included), to clarify the significance, if any, of the wide range of variability we noted in the spore ornamentation of these two taxa. Based on available evidence, however, we do not believe it would be reasonable to treat Isoetes adspersa as separate from I. longissima.

In addition, on the basis of the material we examined in the Braun herbarium (B), we believe I. adspersa represents a later heterotypic (and perhaps even homotypic) synonym of I. capillacea (see above). We could not find original material in P or in ANG.

= Isoetes velata

Observations made on specimens collected in the locus classicus (étang de Richaudron, commune d'Azat [Haute-Vienne, France]) highlight distinctive morphological characters (compared to Isoetes longissima) such as the plant having leaves of reduced size (usually (4-)8-10 cm long vs. (5-)15-35 cm) and thickness (ca. 0.5 mm at mid-length vs. 1-1,5 mm), with a very small margin at the base.

Megaspores (Fig. 1) are significantly different from those illustrated in Berthet & Lecocq (1977), but the specimens they used were not from the locus classicus. Spores are not particularly different from those of Isoetes longissima, although in the distal hemisphere of the megaspore tubercles are more isolated and scattered.

Isoetes tenuissima can also be distinguished from I. longissima by its ecology, typically growing in a completely submerged habitat [START_REF] Prelli | Les Fougères et plantes alliées de France et d'Europe occidentale[END_REF] vs. the seasonally dry condition of the latter. We believe the above described differences in leaf features to be also significant. Finally, the climatic, bioclimatic and biogeographic aspects could support the separation of the temperate I. tenuissima from the mediterranean I. MPU013780, n.v.;PAL No. 18672 !;KFTA No. 0003233, n.v.). -Image of lectotype available at http://coldb.mnhn.fr/catalognumber/mnhn/p/p01266653. Heterotypic synonym of I. tenuissima Boreau [START_REF] Pfeiffer | Monograph of the Isoetaceae[END_REF]Rouhan & Christenhusz, 2017). of that collection, it cannot be considered original material.

Isoetes boryana

At P, in addition to two specimens dated from 1858 with a printed label, we located 10 collections that are part of the original material dated from September 1860.

Many of these specimens were collected on 23 Sept 1860 and distributed through various channels, such as the plant exchange society 'Société Dauphinoise'.

The label of the material distributed by Schultz simultaneously reports two dates, one (3 Sept 1860) is compatible with original material while the other (14 Jul 1861) is more recent than the protologue publication date and is excluded from considerations of the type.

In the protologue, published in June 1861 (Leussink, 1985: 586), the species is said to have been discovered in 1860, and first announced to the public during a meeting of the Société Botanique de France in March 1861 when Durieu presented his new species and distributed some specimens to the members of the Société. See also the note page 165 of the same 'Bulletin de la Société Botanique de France' in which the protologue appears for additional information on the publication date.

Microspores present one of the most challenging mysteries of this taxon. Some authors described the microspores of Isoetes boryana as either smooth [START_REF] Hy | Note sur les Isoetes amphibies de la France centrale[END_REF], Berthet & Lecocq 1977[START_REF] Rolleri | Caracteres diagnosticos foliares en Isoetes (Pteridophyta, Isoetaceae)[END_REF] or very minutely scabrid (Berthet & Lecoq 1977[START_REF] Jermy | Isoetes L[END_REF] or sub-papillose [START_REF] Rolleri | Caracteres diagnosticos foliares en Isoetes (Pteridophyta, Isoetaceae)[END_REF]). Prelli (2002) and [START_REF] Romero | A morphometric study of three closely related taxa in the European Isoetes velata complex[END_REF], however, reported echinate miscropores, similar to those of I. longissima. Our investigations found only non-echinate microspores (Fig. 1).

The microspore of I. boryana shown in [START_REF] Romero | A morphometric study of three closely related taxa in the European Isoetes velata complex[END_REF] is different, however, from the microspore of I. longissima (the two are shown side by side in the same article). [START_REF] Prada | A new species of Isoetes (Isoetaceae) from Turkey, with a study of microphyll intercellular pectic protuberances and their potential taxonomic value[END_REF] found another character separating Isoetes boryana from I. velata/longissima, i.e. the lack of Intercellular Pectic Protuberances (IPP) in the cells of the translacunar diaphragms of the microphylls. In addition, I. boryana is deemed distinctive by its lack of persistent leaf scales [START_REF] Rolleri | Caracteres diagnosticos foliares en Isoetes (Pteridophyta, Isoetaceae)[END_REF].

Another uncertainty of this taxon is its chromosome number: it was reported to be tetraploid, 2n = 44, according to Prada ('unpubl. data' in Rolleri & Prada 2007), but preliminary investigations with flow cytometry suggest a DNA content corresponding to a diploid level (R.Viane, com. pers.), as in I. longissima. The small megaspore diameter (ca. 450 mm) and size comparable to cytologically confirmed associate taxa (Fig. 1) also argue for diploid cytology in this species.

Pending further studies, on the basis of the morphological features discussed above and especially the dramatically different ecology of this taxon, we believe Isoetes boryana to be distinct from I. longissima. Isoetes boryana is a permanently submerged aquatic species or, when occasionally growing emerged, is found only in saturated soil [START_REF] Prelli | Les Fougères et plantes alliées de France et d'Europe occidentale[END_REF]. Isoetes longissima is 'amphibious', growing in temporary (seasonal) pools and spends some (summer) months dormant and without leaves. This species is known from a single collection. The protologue mentions a specimen, collected by Cosson and stored in the herb. Braun: the specimen cited above as the holotype is the only one in B fitting the protologue. Specimen P00466543 in P, which has been labelled as the 'holotype', and the specimen in the Gray herbarium mentioned as 'type' by [START_REF] Pfeiffer | Monograph of the Isoetaceae[END_REF] are in fact isotypes.

Isoetes perralderiana

Our SEM imagery (Fig. 1) highlights a peculiar ornamentation of the megaspores, especially in regards to the distal hemisphere ornamentation pattern.

Being known from a single collection and its status not recently confirmed increases the urgency for further data on this species, whether it is extant, and if so, its morphological variability. On the basis of its macro-and micro-morphology as currently understood and its distinctive ecology (a true aquatic, even in mid summer), we consider Isoetes perralderiana to be distinct from I. longissima. It does, however, show some affinity with I. tiguliana. These species share the presence of phyllopodia (black, indurate remains of dead leaves encircling the leaf rosette at its base) and similar habitats (usually seasonally wet or flooded soils); the taxa hitherto analysed in molecular phylogenetics [START_REF] Larsén | Disentangling the phylogeny of Isoetes (Isoetales), using nuclear and plastid data[END_REF][START_REF] Pereira | Origin and biogeography of the ancient genus Isoëtes with focus on the Neotropics[END_REF] showed conflicting results (two accessions of I. histrix were retrieved in two non-closely related clades, probably due to misidentifications - [START_REF] Larsén | Disentangling the phylogeny of Isoetes (Isoetales), using nuclear and plastid data[END_REF] and/or inaccurate morphological delimitation of the taxa.

Isoetes olympica

Caryological data suggest 2n=20 for I. histrix and 2n=22 for I. gymnocarpa [START_REF] Troia | The genus Isoetes (Isoetaceae): a provisional checklist of the accepted and unresolved taxa[END_REF], but some recent counts suggesting distinction of another taxon similar to I. gymnocarpa with 2n=20 (Bagella & al., 2015) leaves cytological and taxonomic relationships unresolved amongst these taxa. Another specimen (in B) from the same collection is the type of another taxon, Isoetes histrix [unranked] solitaria A.Braun, so far treated as a synonym of I. histrix [START_REF] Troia | A critical conspectus of Italian Isoëtes (Isoetaceae)[END_REF]) but now to be treated as a synonym of I. delalandei.

Isoetes delalandei

Megaspores of I. delalandei (Fig. 2) are almost laevigate, and thus clearly different from the typical megaspores of I. histrix as well as those of I. gymnocarpa (Troia & Greuter, 2015a: 25). Plants of this species are small (about 3-4 cm tall), with arched leaves and abundant phyllopodia exhibiting short lateral teeth.

Isoetes histrix f. subinermis Durieu in Bull. Soc. Bot. France 8: 164. 1861, nom. nud.

No typification has been made, given that the name was not validly published [START_REF] Troia | A critical conspectus of Italian Isoëtes (Isoetaceae)[END_REF]. Because the name has been however widely used in literature and herbaria, we made observations on the unequivocal population noted by Durieu ('bords de l'étang de Cazau' [France]), as an incidental contribution towards disentangling the morphological variation found within the Isoetes histrix group, and towards clarifying the species concept behind that invalid name. Thus, we found that megaspores (Fig. 2) are not tuberculate, as usually seen in I. histrix, but somehow similar to a rugate type (apparently deriving from the fusion of tubercles, and for some aspects resembling the retate type, typical of I. durieui Bory). In the literature it is possible to find SEM images of megaspores of 'I. histrix' s.l. (e.g. Berthet & Lecoq, 1977;[START_REF] Ferrarini | Iconographia Palynologica Pteridophytorum Italiae[END_REF], but unfortunately we don't know to what 'morphotype' they correspond. The only images useful for comparison of this atypical expression with other known morphotypes of I. histrix illustrate polar images of megaspores of I. histrix and I. gymnocarpa collected in Sicily (Troia &al., 2012 andTroia &Greuter, 2015a), and images from the type of I. gymnocarpa in Sardinia (Troia & al., 2015). For comparison purposes, we included in Fig. 2 images of spores from specimens gathered in the (African) locus classicus of I. histrix f. loricata A.Braun representing 'true' I. histrix (s. str.).

CONCLUSIONS

Our morphological observations on the types or on original material or material from the 'locus classicus' help to clarify our knowledge of these two species complex. Now that the morphological and nomenclatural framework is clearer, genetic investigations could significantly further clarify the taxonomic significance of each of the taxa identified here. Further updating of knowledge of the distribution, ecology, and morphological variability of these taxa, particularly with the benefit of fresh material, will be especially useful to this endeavor. In summary, in the Isoetes longissima group, while I. boryana and I. olympica seem deserving of species rank, I. adspersa (better treated as a synonym of I. capillacea) seems indistinct from I. longissima. It is more difficult to decide upon a taxonomic rank for I. tenuissima and I. perralderiana, which we propose to treat as separate species pending further investigations. It is important to note that I. longissima is amphibious, spending a portion of its annual cycle submerged, then becoming emergent, and finally becoming leafless and dormant after its habitat has completely dried up. In marked contrast, both I. boryana and I. tenuissima (and perhaps I. perralderiana as well) are permanent aquatics, most plants being submerged even during the summer, though some individuals can remain non-dormant as emergent on saturated soils.

In the Isoetes histrix group, the possibility exists that I. delalandei and the plants treated under I. histrix f. subinermis nom. nud. represent two taxa different from each other and both distinct from I. histrix. For the present, we accept I. delalandei as a distinct species. Further dedicated and multidisciplinary macro-and micromorphological, ecological, and molecular investigations are needed. These need to be applied to all the other taxa described in this group.

ACKNOWLEDGEMENT

  Taxa considered in this group include Isoetes boryana, I. olympica A.Braun, I. tiguliana Gennari, I. asturicensis (M.Laínz) M.Laínz, I. fluitans M.I.Romero, and I. longissima with its currently recognized five subspecies I. longissima subsp. longissima, I. longissima subsp. adspersa (A.Braun) Troia & Greuter, I. longissima subsp. intermedia (Trab.) Troia & Greuter, I. longissima subsp. perralderiana (Milde) Troia & Greuter, I. longissima subsp. tenuissima (Boreau) Troia & Greuter (Raab-Straube &

  Bory in Compt. Rend. Hebd. Séances Acad. Sci. 18: 1165. 1844. ≡ Isoetes velata var. longissima (Bory) A.Braun in Bory & Durieu, Expl. Sci. Algérie, Atlas: tab. 37, fig. 2. 1849. ≡ Isoetes velata subsp. longissima (Bory) Greuter & Burdet, Med-Checklist 1: 5. 1984. -Lectotype (designated by Troia & Greuter in Pl. Biosyst. 148: 15. 2014): [ALGERIA] 'La Calle, fond d'une Mare dans les Forets pres du Lac Houbéira', 31 May 1841, M.C. Durieu ex herb. Bory St Vincent s.n. (P barcode P00466542!; isolectotype: P barcode P00466541! p.p., detached leaves on the right) -Image of lectotype available at https://science.mnhn.fr/institution/mnhn/collection/p/item/p00466542. = Isoetes setacea var. peyrremondii Bory in Compt. Rend. Hebd. Séances Acad. Sci. 18(26): 1165. 1844 -Type: not designated. The protologue of the name Isoetes setacea var. peyrremondii is supposed to have been published in late June or on July 1 st 1844, based on issue 26 of the 18 th tome of the 'Compt. Rend.…' having already been printed on July 1 st , according to page 50 of the following issue (Compt. Rend. Hebd. Séances Acad. Sci. 19). Among the material present in P, we could not locate any specimen that fits both the date and description of the protologue ('recueillie par M. le capitaine Durieu au bord des flaques d'eau des champs de la plaine d'Oran, où elle persiste jusqu'en mai...' [Algeria]) and belonging to the Bory's herbarium. One possible candidate is P01224865 ('Isoetes lineolata n. sp. Is. setacea b Perreymondii Bory. Algerie. Oran. Flaque desséchée de la plaine, 5 Juin 1844') but it is excluded as potential original material by a note on the upper border of the label ('portée le 20 oct 1844 à la maison par Durieu'= brought to the house on 20 th

=

  Isoetes adspersa A. Braun in Bory & Durieu, Expl. Sci. Algérie, Atlas: t. 37, fig. 3. 1849 -Lectotype (designated here): [illustration] tab. 37, fig. 3, in Bory & Durieu, Expl. Sci. Algérie, Atlas, 1849. -Epitype (designated here): [ALGERIA] mares de la plaine aux environs d'Oran, 5 Juin 1844, (M.C. Durieu) (B barcode B 20 0096547! isoepitypes: B barcode B 20 0096544! P barcode P01224865!other original material: B barcode B 20 0096550!). -Image of lectotype available at http://bibdigital.rjb.csic.es/ing/Libro.php?Libro=3962&Pagina=39; image of epitype available at http://herbarium.bgbm.org/object/B200096547.

  [unranked] intermedia Trab. in Battandier & Trabut, Fl. Algérie Tunisie: 407. 1905 -Holotype: [ALGERIA] Dans une mare au-dessus de Bou-Sfer, 30 Mai 1890, J.B.E. Clary (MPU008563 [image!]). -Image of type available at https://science.mnhn.fr/institution/um/collection/mpu/item/mpu008563. Roux (2009) indicated a specimen collected in Morocco in 1936 ('Morocco. In lacuna oropedii calcarei Atlantis Medii prope castellum Ito, R. Maire & L. Emberger s.n., MPU002740, holo.') as 'holotype' of this name. This actually is the type of another infraspecific taxon, Isoetes velata f. immaculata Emb. & Maire. That same specimen is also erroneously listed by El Oulalidi & al. (2012) as the 'holotype' of I. velata A.Br. subsp. intermedia (Trab.) Maire & Weiller, We did not locate other original material of Isoetes velata [unranked] intermedia, and assumed specimen MPU008563 as to be the holotype. Regardless, we conclude that this name is most probably a heterotypic synonym of I. adspersa A.Braun (= I. capillacea Bory = I. longissima Bory). Dobignard (2017) rightly commented on the difficulty in differentiating the subspecies of I. longissima Bory. Isoetes tenuissima Boreau in Bull. Soc. Industr. Angers 21: 269. 1851. -Type: not designated.

  longissima. Based on this evidence, we tentatively recognize I. tenuissima at the species rank while recognizing the need for further studies. = Isoetes viollaei Hy in J. Bot. (Morot) 7: 432. 1893 -Lectotype (designated here): [FRANCE] (Haute-Vienne) Etang granitique de Riz-Chauvron commune d'Azat, Chaboisseau, September 1857, Hariot, S.E.E.F. 1892, n. 219 (P barcode P01266653!). Remaining syntypes: [FRANCE] Haute-Vienne: étang de Riz-Chauvron, 10 septembre 1863, M.C. Durieu [Flora selecta exsiccata publié par G. Magnier, 1046 bis 'I. tenuissima' Durieu] (MPU barcodes MPU013779,

  Durieu in Bull. Soc. Bot. France 8: 164. 1861 -Lectotype (designated here): [FRANCE] Etang de Cazau à Sanguinet (Landes), 23.9.1860, M.C. Durieu [labelled in Durieu's handwriting] (P barcode P00321072!, one gathering made of 6 individuals at the top half of the sheet); the lectotype is currently mounted on the same sheet with P00321071 (4 individuals only, at the bottom half of the sheet). Isolectotypes: B barcodes B 20 0107144!, B 20 0107155!; P barcodes P00202858!, P00321070!, P00334209!, P01250372!, P01255124!, P01268425!, P01297113!, P01302029!, P06141802!; PAL No. 18589! -Image of lectotype available at http://coldb.mnhn.fr/catalognumber/mnhn/p/p00321072. The plants used by Durieu to describe his new species are in Paris (P). Pfeiffer (1922: 116) cited as 'type' some herbarium sheets stored in MO (barcode 1164548) and US (barcode 01100849), cited as: 'Etang de Cazau, Landes, 7 September, 1863, Durieu (Mo. Bot. Gard. Herb, and U.S. Nat. Herb.)'. In view of the post-publication date (1863)

  Durieu & LeTourn. ex Milde, Fil. Eur.: 282. 1867 -Holotype: [ALGERIA] Sub aqua crescens in stagnulo aquis a fonte Aïn Sumta fluentibus semper replete, infra fauces Akfadou, ad occidentem Urbis Bougie Kabyliae orientalis, circiter ad 1250 m. alt., 1 August 1861, E. Cosson (B barcode B 20 0108215, specimen b!). Isotypes: B barcodes B 20 0108215 a!, B 20 0108214!, FI!, P barcodes P00466543!, P01268221!, W barcode W0000512 (image!), GOET barcode GOET008812 (image!), G barcodes G00349116 (n.v.), G00349117 (n.v.), BM barcode BM001176369 (n.v.). -Image of holotype available at http://herbarium.bgbm.org/object/B200108215_b.

  A.Braun in Milde, Fil. Eur.: 285. 1867 -Holotype: [TURKEY] In kleinen Lachen auf dem Granitplateau des Olymps bei Brussa in Bithynien, ca. 1800 m, 22 June 1866, K. von Fritsch (B barcode B 20 0108203!). -Image of holotype available at http://herbarium.bgbm.org/object/B200108203 Isoetes histrix group This group includes Isoetes histrix, I. gymnocarpa (Gennari) A.Braun, I. sicula Tod., I. delalandei J.Lloyd, I. subinermis (Gennari) Cesca & Peruzzi, I. chaeturetii Mendes. Only I. delalandei remains untypified.

  J. Lloyd, Notes fl. ouest France: 28-30. 1852 -Lectotype (designated here): [FRANCE] île de Houat (Morbihan), April 1852, J. Lloyd (ANG, herb. James Lloyd) (image!); isolectotypes: B barcode B 20 0107589!, NTM! (herb. Toussaint, herb. Menier), P barcodes P01313472!, P01293646!, PC0731960! The publication date of this species name, contrary to what is reported by several sources (including Stafleu & Cowan, 1981) is likely not 1851 but 1852, according to the Lloyd's comments in the protologue. It is also possible to read 'avril 1852' at the end of the protologue, page 30 of the 'Notes'. As it is possible to deduce by reading the protologue, Lloyd received only two specimens (collected in May 1850 in the island of Houat) from Delalande, but given the description including precise information of the habitat, he needed to see more specimens and their habitats before describing the new species; for that, he waited until April 1852 when he was able to personally visit the sites. Accordingly, we consider plants collected in April 1852 to be part of the original material set. Other original material consists of plants collected in May 1850 by Delalande. Those plants and other collected in April 1852 in Belle-Ile are preserved in ANG.