Christophe Petit
email: christophe.petit@unilim.fr

Armelle Chabot

Alexandra Destree

Christiane Raab

A Destrée

R Botella

W G Buttlar

G Graziani

F Hammoum

M Hun

A Millien

TG3 leader G Tebaldi

A Toumi

A Turatsinze

G Airey

B Behnia

A Cannone-Falchetto

O Chupin

A Collop

TG1 leader E Dave

H Di Benedetto

F Dubois

A Graziani

J Grenfell

P Hornych

N Kringos

A Loizos

P Marsac

F Moreno

M L Nguyen

J Pais

C Sauzeat

J M Simonin

Recommendation of RILEM TC 241-MCD on Interface Debonding Testing in Pavements

Keywords: Debonding, interface, pavement, interlayer bond tests

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Scope

Performance and durability of multi-layered pavements strongly depend on the bond between the individual layers and due to stress concentration, a solid bond between surface layers is extremely crucial. Further, there are a number of pavement types with layers constructed of different materials for which the quality of the interlayer bond has been determined essential for avoiding premature distresses such as corrugation, peeling, slippage or fatigue cracking. This recommendation paper lists the most interesting bond testing methods with their main related advises. According to some of these procedures that are mostly standard methods, practical recommendations in term of temperature condition, specimens' size, number of specimen, etc are given. In a last part recommendations for further developments and research are listed.

Lists of abbreviations

Main factors influencing interlayer strength

A first step for more durable pavements is to improve the mechanical performance of interfaces between surface layer and base layer, which are highly submitted to various normal and shear stresses (Figure 1).

During recent decades, in order to try to give answers of how to determine and improve bonding or modified performance, many interlayer bond tests have been developed in various loading conditions [START_REF] Brown | The influence of bonding between bituminous layers[END_REF][START_REF] Vanelstraete | State-of-the-Art Report of RILEM Technical Committee 157 PRC, Systems to prevent Reflective Cracking in Pavements[END_REF][START_REF] Pouteau | Fatigue test and mechanical study of adhesion between concrete and asphalt[END_REF][START_REF] Diakhaté | Shear fatigue behavior of tack coats in pavements[END_REF][START_REF] Piber | RILEM Interlaboratory Test on Interlayer Bonding of Asphalt Pavements[END_REF][START_REF] Raab | Evaluation of Interlayer Shear Bond Devices for Asphalt Pavements[END_REF][START_REF] Partl | Advances in Interlaboratory Testing and Evaluation of Bituminous Materials[END_REF][START_REF] Destrée | Field study to investigate the impact of conditions of application of tack coats on the interlayer bond strength[END_REF][START_REF] Destrée | Field study to evaluate different prenormative interlayer adhesion tests[END_REF][START_REF]Proc. of the 8th RILEM International Conference on Mechanisms of Cracking and Debonding in Pavements[END_REF]. However, interface behaviour between layers is not yet well understood. To investigate the mechanisms of debonding that often occur in pavement structures, different parameters have to be considered [START_REF] Petit | Interface debonding behaviour[END_REF] [START_REF] Chabot | Mechanisms of Cracking and Debonding in Pavements: debonding mechanisms in various interfaces between layers[END_REF][START_REF] Chabot | A 4pt bending bond test approach to evaluate water effect in a composite beam[END_REF][START_REF] Destrée | Impact of tack coat application conditions on the interlayer bond strength[END_REF][START_REF] Ktari | Surface roughness: a key parameter in pavement interface design[END_REF][START_REF] Mateos | Mechanical characterisation of concrete-asphalt interface in bonded concrete overlays of asphalt pavements[END_REF][START_REF] Raab | Cracking and interlayer bonding performance of reinforced asphalt pavements[END_REF].

(a) (b)

Figure 1: Pavement interface fracture debonding modes (a) far from a vertical crack [START_REF] Raab | Interlayer shear performance: experience with different pavement structures[END_REF]; (b) at the edge of a vertical crack [START_REF] Chabot | A modeling to understand where a vertical crak can propagate in pavements[END_REF] Obviously, the mechanical behavior of the pavement materials such as the effect of the viscoelasticity linked to temperature, traffic speed and load (which has not to be considered in a simple way in terms of frequency-dependence conditions for laboratory tests) may have an influence on the interlayer bonding.

Further, environmental effects such as temperature and moisture content of the materials need to be taken into account. In this context, water may play a huge role for interface bonding performance and pavement life, especially when layers show cracks and deficiencies. This fact has been demonstrated for concrete pavements (Figure 2a) [START_REF] Blpc | Concrete pavements : problems raised by the presence of water in their structure (Chaussées en béton : problèmes posés par la présence d'eau dans leur structure)[END_REF][START_REF] Fuchs | Le phénomène du "Punch-Out" sur les autoroutes belges en béton armé continu -causes, effets et remèdes[END_REF] but it is also true for asphalt pavements (Figure 2b) [START_REF] Bergeron | Réparation des nids-de-poule. Info DLC[END_REF]. (a) (b)

Figure 2: Water propagation in pavements from the top and its effect on interface bonding (a)

(from [START_REF] Fuchs | Le phénomène du "Punch-Out" sur les autoroutes belges en béton armé continu -causes, effets et remèdes[END_REF]); (b) (from [START_REF] Bergeron | Réparation des nids-de-poule. Info DLC[END_REF])

Physical interface conditions such as cleanliness and roughness conditions are another important aspect and may lead to very various interlocking situations.

When a tack coat is applied, its intrinsic characteristics (viscosity, breaking index, binder content and the characteristics of the residual binder) play an important role for the adhesion between layers and their type and conditions of application such as application rate, curing time or protection technique are equally crucial [START_REF] Destrée | Field study to investigate the impact of conditions of application of tack coats on the interlayer bond strength[END_REF] [START_REF] Destrée | Impact of tack coat application conditions on the interlayer bond strength[END_REF].

The size of the specimens for laboratory testing has to be taken into account in the interpretation of the results and as stated by many researchers a large number of specimens is needed before final conclusions can be made (see for example [START_REF] Felice | Statistical investigation of two different interlayer shear test methods[END_REF]).

A challenge for design tools regarding the new developments is to include thin surface layer damage and/or structural discontinuities. … Although, the influence of certain parameters is still under debate and different opinions of researchers and national requirements exist, during recent years a common knowledge base on interlayer bonding has been established [START_REF] Piber | RILEM Interlaboratory Test on Interlayer Bonding of Asphalt Pavements[END_REF]. For laboratory testing there are already some existing technics, that enable the user to get more accurate results and to deepen the knowledge of debonding mechanisms such as the used of Digital Image Correlation (DIC) technics. The task group 2 (TG2) of the RILEM TC241 -MCD has proposed to class the existing interlayer adhesion tests into four categories:

• Mode I: Opening mode

Mode I: Opening mode

Interface Mode I opening mode is a debonding or an interface damage phenomenon created by means of a tension loading. Two main types of Mode I tension loading devices have been developed: Tensile Bond Tests (TBT), without notch and Tensile Notch Bond Tests (TNBT) with initial notch. [START_REF] Ktari | Surface roughness: a key parameter in pavement interface design[END_REF][START_REF]Bituminous mixtures -Test methods for hot mix asphalt -Part 48: Interlayer Bonding[END_REF][START_REF] Brrc | Tensile adhesion test[END_REF][START_REF][END_REF]: These tests use specimens without notch and can be applied to assess the capability of tack coats as well as the internal cohesion of the two involved pavement layers. The TBT can be carried out both in situ and in laboratory. These devices mainly differ in the specimen's geometry (cubical -size between 80x80 mm and 100x100mm -or cylindrical -maximum diameter 150 mm), tensile rate (in mm/min or N/s) and the way the test specimens are held (gluing, gripping or clamping). The standard test is usually stress-controlled by applying a tensile force (maximum 200 N/s) until failure. In these standard methods, the estimation of tensile bond strength in obtained by help of three specimens for thin wearing courses (thickness ≥ 30 mm) while six specimens are needed for ultra-thin wearing courses (thickness ≤ 20 mm). For cubical specimens, the tensile load is applied in a low displacement-controlled mode until the specimen fails.

The Tensile Bond Test (TBT)

-Care needs to be taken regarding the adhesive (glue) used to hold the specimen in the tensile device; it should be suitable to avoid failure in the adhesive itself. Here, two-component epoxy glues are recommended.

-Due to the geometry of the tested specimens (cylindrical or cubical), the difficulty of the test is to get uniform tensile stress distribution close to the interface. Shear stresses that exist at the edge of the specimens may affect the results of this "pure" mode I test. The use of specimens with large dimensions as average minimum of six times the maximum size of aggregate is highly recommended.

-The visual assessment of the specimen after the failure is important. The existing modes of failure can be divided into three categories: cohesive, adhesive or mixed.

-For this kind of test failure does not necessarily occur at the interface because debonding happens at the weakest point which can be within the pavement layers, which is one of the main disadvantages of the testing method. So, the most important limitation of the tensile bond tests is that, if the interlayer bond resistance is higher than the in-layer tensile resistance of the two involved specimen's layers, the test result is lower than the actual interlayer bond strength.

-For tests on specimens prepared in the laboratory, the cubical geometry turns out to be more advantageous for DIC analysis than the cylindrical geometry. However, for tests on samples taken on site, a cubical geometry will cause more damage and subsequently extensive repairs.

-In test method allows situ testing, that means the determination of bond strength directly on site. Before applying the gripper system on the pavement surface and applying a tensile force, a ring-groove is generally made beneath the top layer. This has the advantage that if the bond strength is sufficient (no failure up to a maximum force), the test can be done with limited damage to the pavement layers (only the circular groove formed by the coring remains). Although, in case of failure, the different holes obtained may result in more repairing costs and efforts in comparison with the reparation of a "conventional coring" performed on a greater thickness.

-In comparison with temperature-controlled laboratory conditions, the ambient temperature on site will notably influence the test leading to less precise results. However, for some devices such as LAMI, a correction factor that takes into account the test temperature can be applied in case of on site testing.

The Tensile Notch Bond Test (TNBT) [START_REF] Tschegg | Equipment and appropriate specimen shapes for tests to measure fracture values[END_REF][START_REF] Brühwiler | The Wedge splitting test, a new method of performing stable fracture mechanics tests[END_REF][START_REF]Standard Test Method for Determining Fracture Energy of Asphalt-Aggregate Mixtures Using the Disk-Shaped Compact Tension Geometry[END_REF][START_REF] Lugmayr | Mechanism of fatigue crack propagation and fracture behavior in bituminous roads[END_REF][START_REF] Hakimzadeh | Development of fracture-energy based interface bond test for asphalt concrete[END_REF][START_REF] Gharbi | Characterisation of the bond between asphalt layers and glass fiber grid with help of a Wedge Splitting Test[END_REF] This tests use specimens with an initial notch at the interface between layers with the aim to localize and evaluate its fracture properties. The principle of the TNBT consists on opening a crack in double-layered specimen until a complete separation of the two layers takes place. The devices mainly differ in the specimen's geometry (cut straight faces from cores or cubic specimens to facilitate the use of DIC technics), the size and width of the notch, the magnitude and the modal distribution of loads (by means of glued plates, holes, wedge, …) on the two involved specimens' layers.

With TNBT devices, the debonding propagation is obtained in a stable way. In static conditions, tests are usually done under displacement-controlled condition (generally less than 2 mm/min). Tests under water bath are also possible. The obtained results are mostly a load -crack opening displacement curve from which different fracture information can be determined: the maximum tensile stress before the debonding; the fracture energy value; the slope of the decreasing curve that gives the fracture propagation behavior (brittle or ductile) of the interface. Depending on the devices, these tests can be used for fatigue testing of bi-layered specimens.

-For this test specimens' preparation is more difficult and cumbersome than for other tests such as TBT or SBT. Therefore, it is absolutely necessary to pay attention to both the specimen and the notch preparation.

-A large number of specimens must be tested per type of thermal and loading conditions (no less than 10 tests) have to be conducted before concluding on results in terms of fracture energy.

-Compared to tests on cores, the TNBT test offers the possibility to separate large interface planes which may be very convenient for testing the bond between grids (with large meshes) and asphalt. In order to precise the debonding results, it is recommended to evaluate the dead weight contribution of specimens with large dimensions.

Mode II: In plane shear mode

Interface Mode II in plane shear mode is a debonding or an interface damage phenomenon created by means of a shear loading. The shear tests are inspired by shear testing in soil mechanics. It is a well-known fact that the shear test suffers from insufficiencies, since the specimen is subjected not only to shear, but a bending moment is introduced due to an eccentric load induction. Many different types of shear test equipment have been developed in different countries and they can be classified into two categories: pure direct shear tests (without normal stress) and direct shear tests (with normal stress).

In order to introduce the effect of traffic loading some shear testing devices allow the application of a normal load. Equipment with (AST, ASTRA, LISST) or without (Leutner, LPDS, LCB) normal stress are used.

The Shear bond test (SBT) without normal stress [2, 8-10, 25, 34-40] In general, the SBT is a guillotine type test where the shear force is induced directly at one side and not induced at the front surface of the specimen. The test is mostly done on cylindrical specimens extracted from the pavement or prepared in laboratory (LPDS, LCB,…). Mainly two diameters are usually used 100 mm and 150 mm. Otherwise the Double Shear Test (DST) is done on parallelepipedic samples. As for the influence of the diameter, it is found that the maximum shear stress values for all temperatures tested with cores of 150 mm diameter are lower than the results of the test carried out with 100 mm asphalt cores. The direct shear testing devices can be divided into devices which use a clamping or fitting system to hold the test specimen [START_REF] Partl | Shear Adhesion between Top Layers of Fresh Asphalt Pavements in Switzerland[END_REF][START_REF] West | Evaluation of bond strength between pavement layers[END_REF][START_REF] Romanoschi | The characterization of pavement layer interfaces[END_REF] and devices which use a bending mechanism (three or four point shear tests) to apply the shearing [START_REF] Mirò | Evaluation of the effect of tack coats[END_REF]. The gap width between the shearing rings is an important variable, which cannot be neglected. For the test conducted by displacement controlled at a rate of (50 ± 2) mm/min, the European prenorm [START_REF]Bituminous mixtures -Test methods for hot mix asphalt -Part 48: Interlayer Bonding[END_REF] recommends a gap width between the shearing rings ≤ 5 mm). The obtained result is a shear force versus shear displacement graph that allows the calculation of the maximum shear stress, the shear stiffness modulus and the determination of the shear energy. The latest is determined from the area below the shear force-deflection graph as measured between the displacement at maximum shear stress and the intersection of the linear shear force slope with the displacement-axis in (in Nm). The DST allows the investigation of interface behavior in shear monotonic, cyclic and fatigue loading, under controlled temperature and frequency conditions. Fatigue law parameters can be identified [START_REF] Diakhaté | Shear fatigue behavior of tack coats in pavements[END_REF][START_REF] Piber | RILEM Interlaboratory Test on Interlayer Bonding of Asphalt Pavements[END_REF][START_REF] Petit | Pavement Design for Curved Road Sections[END_REF].

The Shear bond test (SBT) with normal stress [START_REF] Raab | Development of a Framework for Standardisation of Interlayer Bond of Asphalt Pavements[END_REF][START_REF] Mohammad | Optimization of tack coat for HMA placement[END_REF][START_REF] Zofka | Advanced Shear Tester for Evaluation of Asphalt Concrete Under Constant Normal Stiffness Conditions[END_REF] Many researchers argue that the normal stress, representing the wheel load on the road, has to be included in interlayer bond testing. Regarding its influence (e.g. the magnitude of normal stress) different opinions and findings are being discussed [START_REF] Uzan | Investigation of adhesion properties between asphaltic concrete layers[END_REF][START_REF] Romanoschi | The characterization of pavement layer interfaces[END_REF]. The application and influence of normal stress is one of the issues, which have been under debate for quite some time. Many of the current designs for shear strength testing equipment produce highly variable results or require complex bi-axial load frames. The so-called LISST uses cylindrical specimens of either 100 mm or 150 mm diameter with a constant rate of 2.54 mm/min, a normal load and possible normal forces. The gap between the shearing and the reaction frame measures 12.7 mm. In the current AST version, specimens of 150 mm diameter are tested. The gap size can vary incrementally between 5 and 12 mm, which may influence the results [START_REF] Raab | Evaluation of Interlayer Shear Bond Devices for Asphalt Pavements[END_REF]. The system allows for various combinations of the static and cyclic loading conditions in the load-or displacement-control mode to be applied to specimens across various temperature conditions.

-Standard tests (SBT) such as Leutner enable the user to determine shear bond performance at a predefined plane and they are very easy to conduct. SBT is a valid method for quality control, but only investigates interlayer bond properties at failure. Further, the testing method does often not consider the application of a normal stress as represented by tire forces on the road. When looking at the interlayer bond properties during the service life of a pavement cyclic testing is recommended. Here, the link between bonding properties and changing temperature effects, rest periods or healing phenomena can be evaluated. Cyclic shear bond results can further be used for interlayer bond performance prediction and modeling purposes.

-Regarding specimens size, greater core diameters of 150mm or preferable to 100mm especially for larger maximum aggregate sizes.

-Gap width is very important: A big gap width will lead to an increased eccentricity resulting in a combined bending-shear stress situation and greater scattering of results. While on the other hand the gap width has to be big enough to account for layers with bigger maximum aggregate sizes and layers with evenness irregularities. Although, European standards allow a gap width between 0 and 5mm, research [START_REF] Raab | Temperature dependency of interlayer shear testing[END_REF]) revealed that a gap width between the shearing rings of ca. 3 mm seems to be optimal regarding the above mentioned criteria.

-Tests conducted in different laboratories have also shown that, for especially for certain constructions such as ultra-thin top layer, a minimum of 6 specimens should be taken for the determination of the average shear bond strength [START_REF] Piber | RILEM Interlaboratory Test on Interlayer Bonding of Asphalt Pavements[END_REF][START_REF] Raab | Methoden zur Beurteilung des Schichtenverbunds von Asphaltbelägen[END_REF][START_REF] Destrée | Evaluation of tack coat performance for thin and ultra-thin asphalt pavements[END_REF].

Mode III: Out of plane shear mode

Torque testing is another way to determine the bond between pavement layers and it can be carried out both in situ and in laboratory [START_REF] Raab | Development of a Framework for Standardisation of Interlayer Bond of Asphalt Pavements[END_REF][START_REF] West | Evaluation of bond strength between pavement layers[END_REF][START_REF] Zahw | Development of testing framework for evaluation of rutting resistance of asphalt mixes[END_REF][START_REF] Choi | A comparison between interface properties measured using the Leutner test and the torque test[END_REF]. Torque testing allows the determination of the torque bond strength between a top layer and the bottom layer. Depending on the working method, the torque is applied until failure occurs or a maximum torque is exceeded. Torque test devices are quite similar compared to one another. In principle, they consist of a gripper system that is glued or fixed to the test specimen and a torque cell applying the torque moment. Torque devices mainly differ in the way the test specimens are held (gluing, gripping or clamping) and in the construction of the test frame (manual or automatic torque devices). The test specimens for laboratory testing are generally cores with diameters of 100 mm, 150 mm or 200 mm (depending on the top layer thickness or the torque test device).

-Torque testing provides a non-uniform shear stress distribution, varying between zero in the center of the interface cross section to a maximum value at its outside.

-Unlike the direct shear testing, the torque testing induces shear stresses at the interface as well as in the surface course of the specimen. The torsion test is therefore less precise than the shear test and also more difficult to interpret in terms of type of failure, since it induces both a torque at the interface and in the upper layer itself, unlike the shear test which concentrates the shear loading exclusively on the interface [START_REF] Destrée | Evaluation of tack coat performance for thin and ultra-thin asphalt pavements[END_REF].

-Since specimens have to be fixed in the torque devices (gluing, gripping or clamping) special care should be taken regarding the fixing mechanism:

• Adhesives (glue) should be suitable to avoid adhesive failure (partly or completely in the adhesive). Here, again two component epoxy glues are recommended. • Gripper systems should hold the specimen strongly and correctly to avoid slippage or deformation of the specimen. -In comparison with the direct shear testing, the torque bond test would be less suitable for laboratory based routine testing of interlayer bond properties because it typically requires fixing the specimen (gluing, gripping or clamping).

-Despite the cumbersome handling, torque testing offers a good possibility for in situ testing and testing of thin surfacing.

-Although in comparison with well-controlled laboratory conditions, the temperature dependence of the torque bond strength on site will notably influence the test result and has to be taken into consideration regarding precision of the test.

-The torque testing has the disadvantage that it is generally performed manually. This requires a certain degree of experience and strength. In fact, it is difficult to keep the application of torque parallel to the interface resulting in axial bending on the specimen and it is sometimes physically difficult to exceed high torque bond strength.

-The weak precision of the manual torque test devices might be a result of the variability and bias introduced by the manual load application. The difficulty of maintaining a constant torque rate manually is one of the reasons why several researchers decided to develop automatic torque devices. The OFTT is one of these automatic in-situ torque devices; it is used in the United States of America to evaluate the long-term post-construction tack coat performance of pavement sections.

-As opposed to the similarity in torque device construction, the torque test protocols generally offer a great variability, which does not simplify the comparison between different tests. This is a fact, which made it impossible to conduct interlaboratory testing in the same manner as for shear or pull-off testing [START_REF] Piber | RILEM Interlaboratory Test on Interlayer Bonding of Asphalt Pavements[END_REF].

-As for the tensile bond test, failure occurs not necessarily at the interface because the debonding happens at the weakest point, which might be within the pavement layers. This means, that if the interlayer bond resistance is higher than the in-layer tensile resistance of the involved interface, the test result does not represent the actual interlayer bond strength.

Mixed Mode: Combination of modes I and II

Some results from the field lead to the conclusion that the interface fracture mode type may not be pure especially near existing vertical cracks through a layer thickness (Figure 1). Three different fracture tests have been proposed by the TG2 researchers of the TC 241-MCD to investigate different questions of interface bonding (behavior) in mixed mode fracture conditions on several type of bimaterial layers [START_REF] Petit | Interface debonding behaviour[END_REF]. First in order to study the durability of thin bonded cement based repairs, a three point bending (static and cyclic) test is used and modelled [47 -48]. This test allows the modelling analysis of the influence of major parameters (such as shrinkage, moisture diffusion, fibre reinforcement, and overlay with low rigidity) on the durability of concrete-concrete bi-layer composites [START_REF] Tran | Delamination of thin bonded cement-based overlays: analytical analysis[END_REF]. Secondly, to investigate the characterization of the crack initiation and propagation at the interface between layers of composite pavements such as UTW, a four-point bending test has been adapted to pavement materials [START_REF] Hun | A four point bending test for the bonding evaluation of composite pavement[END_REF][START_REF] Chabot | Mechanical analysis of a mixed mode debonding test for "composite" pavements[END_REF]. Under displacement controlled conditions, the study of water effect on the bond characterisation, as observed in real roads [START_REF] Barman | Development of design guide for thin and ultra-thin concrete overlays of existing asphalt pavements, task 1 report : Compilation and review of existing performance data and information[END_REF] is possible with help of DIC analysis and a water bath in which the specimens are tested [START_REF] Chabot | A 4pt bending bond test approach to evaluate water effect in a composite beam[END_REF]. Finally, a third test, called a composite specimen interface cracking test (CSIC), has been designed in order to study the optimization of tack coat and application rates [START_REF] Chen | Development of a Composite Specimen Interface Cracking (CSIC) Test for Top-Down Cracking[END_REF]. A hole in the center of the two layers specimen bonded by a tack coat serves both as a stress concentrator to initiate cracking (top-down or reflective) and as a platform for load application. The developed system involves repeated tensile loading and monitoring of number of cycles to failure.

For the evaluation of the performance of the mixed mode tests proposed compared to standard ones, two main following recommendations are given here. It aims to also serve the understanding of debonding mechanisms in pavements.

-The possibility to separate both fracture modes of samples submitted to mixed mode loading is the main objective. At the end, an analysis is expected on the comparison on the results of these mixed-mode tests with the tests conducted in "pure" opening or "pure" shear mode.

-Combined to advanced modelling, advance measurements technics such as the use of DIC technics are a need for separating these different fracture modes.

Conclusions

Interlayer bond testing allows for a great variety of devices and methods. The choice of one or the other does certainly depend on the existing failure mechanism, the purpose of testing (quality control, determination of material property or influencing factor such as moisture, type of tack coat etc.), the situation of testing (in situ or in laboratory), the kind of material tested (thin surfacing, composite material) and many more. In this recommendation paper , practical recommendations for the main standard methods are listed as follows:

-Since shear mode appears to be the most common cause of interlayer bond failure, shear testing (in plane shear mode) -also because of its fairly simple handling and the fact that the shear plane is predefined-is recommended for quality control.

-It has been shown that for interlayer bond testing the amount of specimens should not be too small; for determining mean values in quality control 4 specimens seem reasonable while for research and interlaboratory testing a minimum of 6 specimens is required. -Further, the diameter of test specimens should be preferably 150 mm compared to 100 mm, it has to be at least 6 times the nominal maximum aggregate size. -When looking at the gap width of the shearing rings a size between 2 mm and 3 mm was found suitable. -It is generally known, that interlayer bonding is highly dependent of temperature, test speed and load (static, cyclic, application of normal load) dependent especially when testing asphaltic materials. Therefore, it is absolutely necessary to take these factors into consideration when discussing test results and outcomes. Although, high temperatures are more critical for the debonding of layers, standard test should be performed at a medium temperature of 20°C. -In situ testing is done preferably using torque or pull-off testing. Here, temperature dependency has to taken into account. These two types of test also offer a great potential for interlayer bond testing of thin surfacing. -Due to the gluing For the evaluation of material properties and interlayer bond characteristics cyclic testing which does not only evaluate interlayer bond at failure is recommended.

Regarding the further development of interlayer bond testing, it is crucial to improve the phenomenological understanding of the mechanisms and influencing parameters in interlayer bond testing [START_REF] Chabot | Mechanisms of Cracking and Debonding in Pavements: debonding mechanisms in various interfaces between layers[END_REF].

According to the State of Art Review conducted by the TG2 of the Rilem TC241 MCD [START_REF] Petit | Interface debonding behaviour[END_REF], various deficiencies for studying different parameters have been highlighted. Therefore, future developments and research needs are recommended below. 1. Moisture /water effect infiltration through cracks and other pavement defects have a big influence on the quality of interlayer bond. Moisture was found to reduce the bond strength drastically [START_REF] Chabot | A 4pt bending bond test approach to evaluate water effect in a composite beam[END_REF][START_REF] Mateos | Mechanical characterisation of concrete-asphalt interface in bonded concrete overlays of asphalt pavements[END_REF][START_REF] Partl | Shear Adhesion between Top Layers of Fresh Asphalt Pavements in Switzerland[END_REF]. Its effect should be investigated in more detail and moisture should be integrated in the standard methods and considered in pavement design tools. 2. Need to improve the phenomenological understanding of the mechanisms of interlayer bonding in terms of roughness and the geometrical surface conditions of the interface. In this context the use of model materials [START_REF] Raab | Cracking and interlayer bonding performance of reinforced asphalt pavements[END_REF][START_REF] Raab | Interlayer Bond Testing using a Model Material[END_REF], and finite element modeling as well as roughness measurements at the interface [START_REF] Ktari | Surface roughness: a key parameter in pavement interface design[END_REF] could be very important and useful. These parameters have to be introduced in the standard methods and pavement design tools. 3. Size effect: The specimen size is of great importance for the outcome of interlayer bond testing. As demonstrated by an interlaboratory test program initiated by RILEM [START_REF] Piber | RILEM Interlaboratory Test on Interlayer Bonding of Asphalt Pavements[END_REF] the specimens with smaller diameters were found to achieve higher bond values compared to specimens with smaller diameters. Further, the bigger nominal aggregate size or mesh size (when testing pavements with reinforcing interlayers), the bigger the test specimen should be.

In that case the dead weight contribution of the specimen-might has to be taken into account. 4. Temperature effects have to be studied in more detail. On the one hand shear bond decreases with the increasing temperature, on the other hand increasing temperature may influence the bond positively in terms of healing due to increasing binder viscosity and the compaction effect of traffic. It was further found that interlayer bond defects or weaknesses seem to appear more obvious when testing is performed at very low temperature of -20°C [START_REF] Raab | Temperature dependency of interlayer shear testing[END_REF]. 5. Need to conduct cyclic and static round robin tests to compare the outcome of different devices and methods in more detail and to develop harmonized testing procedures and requirements. 6. Need to compare laboratory results with in situ experiences. Since severe differences between laboratory results and field performance have been identified it appears necessary to define reasons (e.g. ideal laboratory specimens versus real field and construction conditions; laboratory testing versus in situ load application) and modify laboratory testing where required. One way to do so is to test specimens coming from the field and compare the results with those from real scale accelerated loading experiments [START_REF] Gharbi | Characterisation of the bond between asphalt layers and glass fiber grid with help of a Wedge Splitting Test[END_REF]. 7. Need to use energy approaches for modelling interface damage with the aim of quantifying damage evolution during testing. The introduction of rest periods during cyclic will enable the user to assess healing properties and potential. 8. Need to test enough specimens according to the selected type of test and the applied test conditions (for static and cyclic). 9. Need to improve knowledge with advance measurement systems such as:

• NDT (Non Destructive Technics) for field experiment.

• Sensors for the determination of in situ interface bond (shear) values. DIC (Digital Image Correlation) technics for laboratory tests. The local analysis close to the interface area by means of DIC technics. In particular the DIC technics allow the local analysis close to the interface area by means of the measurement of displacements directly above and below the interface. It may help to access the interface mechanical behavior and some of the above parameters [START_REF] Buttlar | Digital image correlation techniques to investigate strain fields and cracking phenomena in asphalt materials[END_REF] facilitating a more accurate analysis of debonding mechanisms and interface behavior.

  • Mode II: In plane shear mode • Mode III: Out of plane shear mode • Mixed Mode: Combination of modes I and II The following paragraphs give the recommendations that can be drawn for each of the proposed interface bond characterization tests that have been collected in the Chapter III of the State of the Art of the RILEM TC241-MCD [1].

Acknowledgments Thanks to O. Abraham, F. Canestrari, F. Fouchal, A. Guarin, K. S. Hakimzadeh, B. Hill, F. Lebon, R.B. Miro, F.E. Pérez-Jiménez, J.M. Piau, B. Picoux, C. Plati, O. Pop, R. Roque, C. Sullivan and M. Takarli for their contributions in the Chapter III " Interface debonding behavior" of the MCD STAR document from which a part of this recommendation has been provided.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


Benoit De Thoisy
email: benoit@kwata.net

Ana Carolina Pavan

Marguerite Delaval

Anne Lavergne

Thomas Luglia

Kévin Pineau

Manuel Ruedi

Vincent Rufray

François M Catzeflis
email: francois.catzeflis@univ-montp2.fr

Kevin Pineau

Kevin Pineau

Cryptic Diversity in Common Mustached Bats Pteronotus cf. parnellii (Mormoopidae) in French Guiana and Brazilian Amapa

Keywords:

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mormoopidae is a widely distributed family of Neotropical insectivorous bats ranging from the southwestern United States to southern Brazil, including many islands in the Antilles. The Mormoopidae is composed of two genera, Mormoops and Pteronotus, and 10 species [START_REF] Simmons | Order Chiroptera[END_REF]. One species, the Common Mustached Bat (Pteronotus parnellii) is the only high-duty cycle echolocating bat of the Neotropics, i.e. one that uses harmonics of Constant-Frequency (CF) calls to orientate themselves [START_REF] Schnitzler | How echolocating bats search and find food[END_REF]. This species was described by [START_REF] Gray | List of the specimens of Mammalia in the collection of the British Museum[END_REF] from animals originating in Jamaica, but it is traditionally viewed as a widespread species living across most of the Antilles, Central and tropical South America (e.g. [START_REF] Simmons | Order Chiroptera[END_REF]. Eight subspecies have been described throughout this wide range, including some former continental species that are now considered as synonyms of P. parnellii (reviewed in [START_REF] Herd | Pteronotus parnellii[END_REF][START_REF] Simmons | Order Chiroptera[END_REF].

However, recent phylogenetic studies revealed that populations of the Common Mustached Bat (CMB) are not homogeneous and bear much hidden biodiversity across their geographic range. Using sequences of the Cytochrome b gene (Cyt b), [START_REF] Lewis Oritt | Molecular systematics of the family Mormoopidae (Chiroptera) based on cytochrome b and recombination activating gene 2 sequences[END_REF] showed that animals from the type locality in Jamaica are genetically very distinct (11 % sequence divergence) from specimens in Suriname or Guyana, while similar levels of divergence separate some bats from Suriname and those from Guyana or Mexico. By using an improved geographic sampling and combining mitochondrial and nuclear genes, [START_REF] Davalos | The geography of diversification in the mormoopids (Chiroptera: Mormoopidae)[END_REF] confirmed the large molecular divergence between P. parnellii sampled in the Antilles (Jamaica, Puerto Rico and Hispaniola), and mainland lineages. One of these divergent lineages was located in French Guiana and Suriname, and the other in Mexico, Honduras and Guyana. Based on genetic, morphological and distributional data, [START_REF] Davalos | The geography of diversification in the mormoopids (Chiroptera: Mormoopidae)[END_REF] and [START_REF] Van Den Bussche | Mitochondrial and nuclear DNA sequence data provide resolution to sistergroup relationships within Pteronotus (Chiroptera: Mormoopidae)[END_REF] proposed that the mainland CMB might represent several cryptic species that are not conspecific with the true Jamaican P. parnellii. [START_REF] Davalos | The geography of diversification in the mormoopids (Chiroptera: Mormoopidae)[END_REF] further suggested that the name P. rubiginosus (Wagner, 1843) would be appropriate to name the continental lineage represented by CMB from French Guiana, Guyana, Suriname, Mexico and Honduras.

More recently, [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF] analyzed mitochondrial and nuclear gene variation together with morphological and some acoustic data to show that the CMB found in Central and South America represent at least three or four independent, biological species, and confirmed that the name P. parnellii should be restricted to bats from Jamaica and perhaps other Greater Antilles islands. These authors further used DNA barcodes of the Cytochrome oxydase 1 gene (CO1) to circumscribe four major clades of continental CMB that may represent each a distinct species. The correlation of these molecular clades with morphological characters was good, but none of the individual measurements were able to discriminate all clades. Using this integrated approach, [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF] reached the following taxonomic conclusions for continental taxa: (i) their clade 1 (or Group 1) is comprised of all CMB sampled in Central America (from Panama to southern Mexico), are relatively small (mean forearm length 59.9 mm), emit CF echolocation calls at about 62 kHz and could be named P. mesoamericanus. (ii) their clade 2 (Group 2) is comprised of CMB sampled in Venezuela, Trinidad and western Guyana, are medium-sized (mean FA 62.4 mm), and emit CF calls at about 59 kHz (although no calls from continental animals could be obtained), and would be an unnamed species (P. sp2). (iii) their clade 3 (Group 3) includes specimens from Guyana and Suriname, are medium-sized (mean FA 63.2 mm), and represent most likely another unnamed species (P. sp3);. (iv) their clade 4 (Group 4) includes specimens from the same area as those of clade 3 (Guyana and Suriname), but are significantly larger (mean FA 65.2 mm), and represent a forth unnamed species (P. sp4). The echolocation calls recorded from free-flying CMB from Guyana had CF at about 53-54 kHz, but as no match with genetically identified animals could be obtained, [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF] were unable to associate these call characteristics to either clade 3 or clade 4.

In this study, we describe the bioacoustic variation of over 200 CMB sampled in French Guiana and nearby Amapa State in Brazil, to show that two distinct phonic types live in sympatry in extended parts of this geographic range. We also characterize the molecular identity of these two phonic types and link them to published samples or clades to reach informed taxonomic recommendations. Morphological characters, although not completely discriminant between the two phonic groups, are also described to provide an aid for their identification in the field or for museum specimens.

Materials and methods

Sampling

Mormoopid bats were sampled in 14 localities of French Guiana and Amapa (Fig. 1), from which we collected data of either or all of bioacoustics, external and cranial morphometry, and molecular systematics.

Capture methods included essentially mist nets set either at the entrance of caves or across nearby corridors such as trails in the forest. Mist nets of 2.6 × 6 m and 2.6 x 10 m were employed at ground level. In addition, we used at Cacao (locality 3 in Fig. 1) a three-frame harp trap (AUSTBAT Research Equipment, Victoria, Australia), with a catching surface of 1.0 m2 erected across a trail acting as a corridor for bats flying out of a nearby cave system.

External measurements and ultrasonic calls were recorded for all captured bats after which they were released in the same spot. A small biopsy punch [START_REF] Worthington-Wilmer | A non-lethal method of tissue sampling for genetic studies of Chiropterans[END_REF] used for DNA analyses was also taken from some individuals prior to release, and a small selection of specimens were preserved as scientific vouchers for further cranial measurements. These vouchered specimens were euthanized, fixed in 10% buffered formalin and finally stored in 70% ethanol. Appendix 1 lists the origin and institutions housing these vouchers.

Morphology

Gender and reproductive status were acquired from external characteristics (e.g. enlarged nipples or testis) or from gross examination of dissected specimens [START_REF] Racey | Reproductive assessment of bats[END_REF]. All specimens reported here were adults as indicated by completely fused phalangeal epiphyses. The following external measurements were taken with a dial caliper accurate to 0.1 mm: forearm length (FA), metacarp of third digit (MC3), metacarp of 4 digit (MC4), total length of fourth digit (D4), hindfoot length excluding claw (HF), tibia length (TI), and ear length from basal-ventral notch of the pinna to its tip (Ea). Digit measurements were taken from the wing held flat on a solid surface and excluding wrist.

Skull and dental measurements were taken with a dial caliper (accurate to 0.05 mm) following the methods detailed in Gutierrez and Molinari (2008) and [START_REF] Simmons | The mammals of Paracou, French Guiana: A Neotropical lowland rainforest fauna. Part 1. Bats[END_REF] except when noted: greatest length of skull (bone-to-bone: GLS) and greatest length of skull including the upper incisors (GLSI), braincase depth (BRD), maxillary toothrow length (MTL), condylo-incisive (CIL) and condylo-canine lengths (CCL), palatal length (distance between the posterior palatal notch and the external border of the incisives PL), length of upper molars (M1M3), zygomatic breadth (ZB), mastoid breadth (MB), breadth across molars (BaM), mandibular toothrow length (MDT), mandibular condylo-canine length (MCC), coronoid height of mandible (CRH). For comparing the French Guianan material with topotypic material of P. rubiginosus, we examined a museum specimen (MZUSP-35152) caught at Cuiabá, Mato Grosso, Brazil. This city is located 180 km east-north-east from Cáceres, which is close to the type-locality of P. rubiginosus defined by [START_REF] Gardner | Mammals of South America, Volume 1. Marsupials, Xenarthrans, Shrews, and Bats[END_REF] as being at 16°04' S, 57°43' W.

To differentiate both phonic types, we performed with Statistica 6.0 (StatSoft Inc., USA) two discriminant function analyses (DFA), one based on three external measurements taken in all 201 recorded bats (FA, MC3 and MC4), and one based on 14 cranial (M1M3, GLS, GLSI, CIL, CCL, ZB, MB, MTL, BaM, CRH, PL, MDT, MCC, BRD) measurements of a subset of 23 vouchered specimens. Each bat was classified a priori according to its recorded phonic type (53 or 59 kHz), except for the Mato Grosso specimen which was left unclassified prior to analyses. To reduce the number of cranial variables entered into the discriminant function, we computed a stepwise discriminant function, where only those first variables contributing the most to the discrimination of the two groups are entered. Univariate statistical tests for comparing measurements or acoustic calls between groups included Mann-Whitney nonparametric test, as implemented by the software PAST [START_REF] Hammer | PAST: Paleontological Statistics Software Package for Education and Data Analysis[END_REF].

Bioacoustics

Echolocation calls were recorded with Pettersson bat detectors (models D240X, D500X, D1000X: Pettersson Elektronik AB, Uppsala, Sweden) and stored using a Roland -R-05 recorder. Calls were analyzed with the program BatSound Pro 3.31 (Pettersson Elektronik AB, Uppsala, Sweden) based on spectrograms with Hanning window at a sampling rate of 44,100 Hz and Fast Fourier Transformation (FFT) size of 512. Part of the sonograms were also created with R 2.14.1 (R Development Core Team, 2011) using the package Seewave [START_REF] Sueur | Rapid Acoustic Survey for Biodiversity Appraisal[END_REF]. For each recorded call the [second] harmonic containing most energy was identified from the power spectrum and measurements taken from the Constant Frequency (CF) component of the call, as detailed in previous studies [START_REF] Schnitzler | How echolocating bats search and find food[END_REF].

with the EasyMag © Biomérieux robot. Two mitochondrial genes were targeted: the Cytochrome b (Cyt b), amplified using the pairs MVZ05/NEW12 and UMMZ04/UMMZ13 primers (Jansa et al. 1999, Dávalos and[START_REF] Davalos | Phylogeny of the Lonchophyllini (Chiroptera: Phyllostomidae)[END_REF], and the Cytochrome oxydase 1 (CO1), amplified with the methods commonly used in barcoding screening [START_REF] Borisenko | DNA barcoding in surveys of small mammal communities: a field study in Suriname[END_REF][START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF]. After amplification, PCR products were sent for purification and sequencing at Cogenics (Takeley, UK). Sequences were aligned and checked manually with MEGA 5.1 [START_REF] Tamura | MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF] for absence of gaps or stop codons, to ensure that these were not pseudogenes. Besides the French Guianan, Amapa and the rubiginosus specimen from Mato Grosso (MZUSP-35152), the Cyt b and CO1 sequences were acquired from three additional specimens caught by B. Lim in Jamaica, for the sake of having comparative materials from near the type-locality of P. parnellii as well. All sequences obtained in this study have been deposited in GenBank and are listed by their accession number in Appendix 1.

Genetic relationships of CO1 haplotypes were visualized on statistical parsimony networks with the minimum connection probability set to 95% [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF].

The phylogenetic relationships between samples were evaluated using maximum likelihood (ML), Neighbor-Joining (NJ), and Bayesian procedures. The GTR model [START_REF] Tavaré | Some Probabilistic and Statistical Problems in the Analysis of DNA Sequences[END_REF], with gamma (G) rate parameter and a proportion of invariant sites (I) was identified with MEGA 5.0 [START_REF] Tamura | MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF] as the best fitting nucleotide substitution model for both genes. Ten thousand replicates were used for maximum likelihood analyses in Mega 5.0, yielding support values of Bootstrap Percentage (BP).

The Bayesian approach was carried out with MrBAYES 3.2 [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF].

Markov Chain Monte Carlo (MCMC) simulations were run twice independently for 10 million generations with four simultaneous chains, using a sample frequency of 1,000 and a burn-in of 2,500 trees; support values are indicated by Posterior Probabilities (PP), and were calculated from the remaining trees. Intra and inter-clade haplotypic and nucleotidic variabilities were calculated with ARLEQUIN 3.5 [START_REF] Excoffier | Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows[END_REF]. Pairwise genetic distances were calculated with MEGA using the K2P model in order to be easily comparable with other similar studies.

For the sake of clarity and for facilitating comparisons with previous works, we indicate in Appendix 2 the names we have used for the sequences retrieved from GenBank, their approximate localities, and the voucher numbers (when known).

Results

1) Bioacoustics

Grotte Mathilde (locality 5; Fig. 1) is a cave surrounded by primary rainforest located ca. 40 km south-east from Cayenne. At this locality, we verified that CMB held individually in cotton-bags emitted ultrasonic calls that had CF characteristics indiscernible from those emitted when flying (upon release). Differences measured for each bat recorded in the bag or upon release were negligible (standard deviation less than 0.2 kHz), which validates the assignation of all handled bats into their respective phonic types.Based on these coustic assignations, 63 CMB caught at Grotte Mathilde emitted ultrasounds with a CF component around 53 kHz (called hereafter the 53 kHz phonic type), and 57 bats had CF components at around 59 kHz (i.e. the 59 kHz phonic type).

We then addressed the population variability of the CF component of both phonic types in samples from Grotte Mathilde (the same 120 individuals), Cacao (87), and , where these phonic types were recorded in sympatry. At those three sites the two distinct phonic types were clearly discernible with no intermediate CF value (Table 1). At any site, the difference between the highest CF value in the 53 kHz phonic type and the lowest in the 59 kHz phonic type was at least 2.3 kHz (Grotte Mathilde), whereas the difference in mean CF values between each phonic type varied between 5.45 kHz (Cacao) and 6.13 kHz (Haute Camopi). Part of the slight differences observed between localities among each phonic type could be due to technical aspects related to the different models of bat-detectors used to record the CF calls, but again these differences were much smaller than those characterizing each phonic type (Table 1).At the regional scale, all individuals from 6 different localities in French Guiana were assigned to their respective phonic type (53 or 59 kHz), and again CF calls showed minimal variability within each group (53.4 + 0.62 kHz, N = 130; 59.2 + 0.68, N = 127; respectively). No overlap was observed between phonic types (Fig. 3). The classification of all handled or free-flying CMB through their ultrasounds thus provides an easy and reliable character for recognizing the two phonic types in French Guiana.

2) Molecular systematics and taxonomy

The CO1 barcode fragment (657 bp) was sequenced in 14 vouchered specimens from the area of sympatry of Grotte Mathilde and whose ultrasonic calls were also recorded. The alignment of sequences resulted in five distinct haplotypes. Two were unique to the six 53 kHz phonic type CMB, and three unique to the eight 59 kHz animals. TCS networks confirm that haplotypes from each phonic type form statistically separate networks (at the 95% threshold), and differ by a mean K2P distance of 4.9 % (Fig. 4). Haplotypes differed by one mutation (0.06 % net distance) and by up to 9 mutations (0.56% net distance) within the 53 and 59 kHz phonic types, respectively

The more global phylogenetic analyses of DNA barcodes (CO1 fragments) included 5 distinct sequences from French Guiana, 2 from Brazilian Amapa, one from Brazilian Mato Grosso, and the unique haplotype found in 3 CMB from Jamaica, as well as 17 published CO1 sequences from various localities in Suriname, Guyana, Venezuela, El Salvador and Mexico [START_REF] Clare | Neotropical bats: estimating species diversity with DNA barcodes[END_REF][START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF]. Phylogenetic analyses indicate that the French Guianan and Amapa bats of each phonic type also segregate in two monophyletic clusters (Fig. 5). Noteworthy, the CO1 sequence of specimen MZUSP-35152 is nested within the cluster containing all CMB identified as Pteronotus sp4 by [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF], with less than 0.1% K2P distance from other sequences in this clade. Each major clade (denoted as P. sp3 and P. sp4) contains animals from various localities of Suriname and Guyana -see Appendix 2 -and both are well supported by high bootstrap and PP values (Fig. 5). These two clades show low intraspecific variability (less than 1.4% K2P) and form closely related, sister taxa differing by a mean of 5.1% K2P distance.They are more distantly related to any other named species of Pteronotus sampled in this analysis.

Also apparent on the phylogenetic tree is a cluster of four CO1 sequences containing CMB from Mexico (ROM-95741), El Salvador (101476), Venezuela (107924), and western Guyana (101046) and corresponding to P. sp1 and P. sp2 of [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF].

This second group is sister to the other species (P. sp3 and P. sp4) from the Guiana Shield. The CO1 haplotype from Jamaica (ROM-120826) representing P. parnellii is external to the clade containing P. sp1 to P. sp4 (Fig. 5) and differs from continental CMB by a mean K2P of 11.4 %.

The Cyt b gene was sequenced from 4 vouchered specimens whose ultrasonic calls were previously recorded at Grotte Mathilde, from one specimen in Cacao, two specimens in Amapa, and one specimen in Mato Grosso (Appendix 1). Additional Cyt b sequences of CMB and other species of Pteronotus [START_REF] Lewis Oritt | Molecular systematics of the family Mormoopidae (Chiroptera) based on cytochrome b and recombination activating gene 2 sequences[END_REF][START_REF] Davalos | The geography of diversification in the mormoopids (Chiroptera: Mormoopidae)[END_REF]) retrieved from Genbank and included in phylogenetic analyses for a total of 32 mormoopid and 4 outgroup taxa (Fig. 6). Details on origins of these sequences are given in Appendix 2.

All sequences of the 59 kHz phonic type bats appear among the putative species P. sp3, while those of the 53 kHz phonic type group within P. sp4, both with strong support (BP of 91 and 99, respectively; 1.0 PP: Fig. 6). The latter putative species also includes the single specimen from near the type-locality of rubiginosus. Average divergence within these two clades was 1.0 and 1.6 % nucleotides, respectively. Each clade included animals caught in sympatry in French Guiana or Amapa, and appear as each other's closest relative (at 6.6 % K2P) in all reconstructions (Fig. 6).

The putative species P. sp2 (from western Guyana) and P. sp1 (Mexico; Honduras) form another pair of closely related taxa and appear in a clade (99 BP, 1.0 PP) which is sister to a well supported group (97 BP, 1.0 PP) containing the Antillan species P. parnellii (Jamaica), P. pusillus (Dominican Republic), and P. portoricensis (Puerto Rico).

All four putative species and the Antillean CMB cluster in a large, well supported clade (99 BP, 1.0 PP) which forms a polytomy together with the seven remaining species of Pteronotus (macleayii, quadridens, fulvus, davyi, gymnonotus, psilotus and personatus).

3) External and cranial morphology

Forearm and third metacarpal measurements tended to be larger in females than in males in both phonic types, but differences were mostly insignificant (Table 2) and hence we pooled genders for other comparisons. Differences among phonic types sampled across French Guiana were in turn all highly significant (Table 3) with individuals of the 53 kHz phonic type being on average larger than those of the 59 kHz phonic type.

Although mean values differ, the ranges of the external measurements overlapped broadly, as illustrated with FA length (Fig. 7).

Similarly, the three external variables (FA, MC3, MC4) retained to best discriminate the two phonic types are insufficient to identify each phonic type with confidence, although the discriminant function analysis (DFA) is highly significant [F(df 3,196) = 67.168, p<0.0001]. Indeed 19 out of 110 individuals identified by their ultrasounds as 59 kHz phonic type and 12 out of 89 identified as 53 kHz phonic type were misclassified by this discriminant function (results not shown). Interestingly, the male individual from Mato Grosso (MZUSP-35132) is classified as a 53 kHz phonic type (posterior probability 0.76) with this simple function. Results were similar if sex were separated or pooled in the discriminant analyses. Clearly none of these external characters taken alone or in combination can be used for a reliable phonic type discrimination.

Cranial and dental measurements also indicate that 53 kHz CMB are larger than 59 kHz bats and, despite small sample sizes, differ significantly from each other (Table 4). Most cranial measurements do not overlap between individuals of the two phonic types (Table 4), but these results should be considered preliminary due to the small number of skulls examined.

A discriminant function analysis including all 14 variables is highly significant [F(df 14, 7) = 20.23, p<0.0003] and classified all 22 skulls in their correct phonic type (results not shown). We present here only results of the stepwise discriminant function [F (df 3, 18) = 64.42, p < 0.0001; Fig. 8] that includes the three most discriminant cranial variables (MDT, BRD and CCL) and which classifies all individuals in their correct (a priori) phonic type. Again the skull of the Mato Grosso Pteronotus (specimen is classified a posteriori as a 53 kHz phonic type in these discriminant analyses. This male individual was thus classified as a 53 kHz phonic type with high posterior probability (>0.76) in all preformed DFA, whether based on external or on cranial measurements.

Discussion

Our results clearly support the existence of two phonic types among CMB living in French Guyana and Brazilian Amapa. Each phonic type emits constant frequency calls at non-overlapping ranges (Fig. 3). Indeed both sexes of the 53 kHz phonic type emit around 53 kHz with very little variation across the region (extremes 51.5-54.5 kHz; Table 1). Likewise, CMB of the 59 kHz phonic type emits at around 59 kHz (range 56.8-60.1 kHz; Table 1) in all recorded localities. These two phonic types are therefore easy to identify throughout their echolocation calls, including when they are recorded when held captive in cotton bags.

The two phonic types are found throughout French Guiana and Brazilian Amapa (Fig. 1), but further bioacoustic studies are needed to understand their exact distribution in this region. Where they are found in sympatry, both phonic types occur in comparable numbers (Table 5), suggesting that they can coexist in similar habitats without major ecological competition. Indeed, they may occupy the few cave roosts existing in this area, while exploiting a different trophic niche of their habitat, as was evidenced in other sympatric, sibling species of bats sharing roosts (e.g. [START_REF] Arlettaz | Trophic resource partitioning and competition between the two sibling bat species Myotis myotis and Myotis blythii[END_REF].

Molecular analyses of two mitochondrial genes (CO1 and Cyt b; Figs 5 and6) consistently identify these two phonic types in the clades defined by [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF].

The sequences of 59 kHz phonic type bats correspond to clade 3, which is considered as a putative species P. sp 3, while the 53 kHz phonic type sequences cluster within clade 4 (P. sp 4). Both phonic types are closely related (mean 6.6% K2P distance for the Cyt b and 5.1% for CO1), sister clades in all phylogenetic reconstructions and present little genetic variation within clades (Figs 5 and6). Although sample sizes of CMB characterized by both bioacoustics and genetic data are still limited (14 individuals), there is complete concordance between phonic types and molecular clades (Fig. 4), indicating that no gene flow occurs between animals of both phonic types, even when they share roosts. Our results therefore fully corroborate the conclusions of [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF] who consider that clades 3 and 4 represent independent, biological species.

Interestingly [START_REF] Clare | Neotropical bats: estimating species diversity with DNA barcodes[END_REF] identified molecularly nearly the same number of P. sp3 (n= 143) and P. sp4 (n= 151) individuals in a large survey (355 barcoded bats) of CMB sampled in Suriname and Guyana, a pattern of relative abundance similar to that found in French Guiana.

Morphologically, these two species differ by size, the 53 kHz phonic type being significantly larger than the 59 kHz type in all external (Table 3) andcranial (Table 4) variables measured. These differences in body size and in echolocation calls parameters are consistent with expectations from the scaling effect of size [START_REF] Jones | Scaling of echolocation call parameters in bats[END_REF], where larger species tend to emit calls at lower frequencies when compared to smaller ones.

Unfortunately, these morphological differences, although highly significant, do not allow for a unambiguous separation of both phonic types, as most external characters show large measurement overlaps (e.g. forearm length, Fig. 7). Clearly, ultrasound recording is a much more reliable method to identify living CMB of both phonic types in French Guiana and Amapa. Individual measurements of the skull are much less overlapping (Table 4) and allow for a complete discrimination of all bats in a simple discriminant analyses (Fig. 8). Skull dimensions are therefore also appropriate to identify dead or museum specimens, e.g. for proper taxonomic comparisons.

In an effort to assign an appropriate taxonomic name to the two cryptic phonic types, we included one CMB (MZUSP35152, see Appendix 1) sampled near the type locality of rubiginosus (Wagner, 1843). This taxon name was suggested by [START_REF] Davalos | The geography of diversification in the mormoopids (Chiroptera: Mormoopidae)[END_REF] for CMB of eastern South America, but because there are clearly more than one species of CMB living in this region, it was unclear if this name would be applicable. In all comparisons the specimen MZUSP35152 unambiguously is part of the 53 kHz phonic type (= P. sp4). Indeed, despite extensive geographic distance separating samples of P. sp4 from French Guiana and the Mato Grosso (over 2'000 km), both mitochondrial markers show little genetic differentiation (Fig. 6). Morphologically the relatively large external measurements of this Mato Grosso CMB also fits the range of variation displayed by CMB of the 53 kHz phonic type in French Guiana (Table 3) and cranially it matches skull dimensions typical of specimens from this phonic type (Fig. 8,Table 4). If the Mato Grosso individual truly represents rubiginosus, then this name would apply to the 53 kHz phonic type (P. sp 4) and would considerably extend its geographic range beyond the Guianan Shield. These characteristics also fit with the brief morphological description of clade 4 given in [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF].

The smaller CMB of the 59 kHz phonic type and representing P. sp3 are more difficult to classify. So far, these animals are found only on the Guianan Shield (French Guiana, Brazilian Amapa, Guyana and Suriname, Clare et al., 2013 and Fig. 1) where no endemic taxon have been named. This phonic type most probably represents a new cryptic species in the CMB complex, as suggested earlier. At a broader geographical scale, a third taxon of CMB denoted P. sp2 and also characterized by constant frequency calls around 59 kHz is found in the north-western part of the Guianan Region (Guyana and the Venezuelan states of Amazonas and Bolivar; [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF]. Representatives of this third cryptic species are genetically very distinct from the 53 and 59 kHz phonic types from French Guiana in all reconstructions (Figs 5 and6) and certainly represents another cryptic species, related to the Central American P. sp1 (= P. mesomaricanus in [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF]. We did not sample P. sp2 in French Guiana or Brazil, and as no precise morphological or bioacoustics clues are provided for its identification, it is also premature to name it properly. Clearly reference material from other type localities of CMB taxa and more specimens characterized with multiple methods are needed before all cryptic species within the CMB complex can be critically reviewed for an adequate systematic arrangement.

The existence of at least three cryptic species of CMB living in the Guianan Shield, some of which may live in strict sympatry and share roosts (Fig. 1) has important bearings for their conservation, as their exact distribution, ecological needs and abundance are largely unknown.

Much remains to be done for getting a complete picture of Pteronotus systematics and biodiversity in Central and South America. Studies should investigate molecular and ultrasonic call characters in Venezuela, where Gutierrez and Molinari (2008) have shown through comparative morphometrics that two taxa live in parapatry, namely P.

(parnellii) fuscus north from the Rio Orinocco and P. (parnellii) rubiginosus south from the Rio Orinocco, i.e. in the north-western part of the Guianan Region. An appealing hypothesis is that P. (parnellii) rubiginosus of Guttierez and Molinari (2008) corresponds to Group 2 (P. species 2) of [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF], i.e. animals from the Guianan Region in western Guyana and in Venezuela (states of Amazonas and Bolivar), for which we here use the taxon name Pteronotus sp2. Gutierrez and Molinari (2008) indicate that the Venezuelan CMB north of the Orinoco (which they call P. parnellii fuscus) have a Constant Frequency echolocation call at around 62 kHz, a value similar to the one reported by [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF] for what those latter authors call Pteronotus sp1 (or P. mesoamericanus). Clearly, more work combining different kinds of characters is needed for understanding those conflicting interpretations, as [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF] circumscribe their P. mesoamericanus CMB to Central America (from Panama to Southern Mexico) whereas Gutierrez and Molinari (2008) and 2). The tree was rooted with sequences of P. personatus and P. gymnonotus.

Specimens (with voucher numbers) were assigned to Pteronotus sp1 to sp4 according to [START_REF] Clare | Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii[END_REF]. The phonic type of the individually recorded bats is also given in the label.

  have their P. fuscus CMB limited to Caribbean regions of Colombia and Venezuela. Table 4: Cranial and dental measurements (in mm) in French Guianan Common Mustached Bats: 8 individuals of the 53 kHz phonic type and 14 individuals of the 59 kHz phonic type. Mann-Whitney tests of differences between the phonic types are all significant at p < 0,001. The far right column is for specimen MZUSP-35152, from Cuiabá, Mato Grosso, Brazil (see text for further details). Abbreviations: GLS = greatest length of skull , GLSI = greatest length of skull including upper incisors, CIL =condyleincisive length, CCL = condylo-canine length, PL = palatal length, ZB = zygomatic breadth, BaM = breadth across molars, MCC = mandibular condyle-canine length, MTL = maxillary toothrow length, M1M3 = length of upper molar teeth, MDT = mandibular toothrow length.

Figure 1 :

 1 Figure 1: Map of French Guiana (localities 1 to 9) and Amapa, Brazil (10 to 14) locations where bats of the 53 kHz (white circles) and the 59 kHz phonic types (black circles) have been characterized through bioacoustics, skull morphometry and/or molecular genetics. Localities harbouring both phonic types in sympatry are indicated by a pie circle. 1 = Macouria River, municipality of Tonate, 05°02'N; 52°30' W 2 = Quartier Torcy, municipality of Roura, 04°50' N; 52°16' W 3 = Cacao, municipality of Roura, 04°34' N; 52°27' W 4 = Trésor Natural Preservation, municipality of Roura, 04°37' N; 52°17' W 5 = Grotte Mathilde, municipality of Regina, 04°31' N; 52°07'W 6 = Montagne des Gouffres, municipality of Regina, 04°20' N; 52°16' W 7 = Saut-Pararé, municipality of Regina, 04°02' N; 52°42' W 8 = Montagne Cacao by Haute Camopi, municipality of Camopi, 02°20' N; 53°12' W 9 = La Trinité, municipality of Saint Elie, 04°61' N ; 53°40' W 10 = Iratapuru village, near to Laranjal do Jari, Jari river, 00°37' N, 51°31' W 11 = Itapeuara village, near to Laranjal do Jari, Jari river, 00°29' N ; 52°41' W 12 = P.N.Tumucumaque, Rio Mutum, municipality of Calçoene, 01º23' N; 51º55' W 13 = P.N.Tumucumaque, Rio Anoteie, municipality of Oiapoque, 03º13' N; 52º01' W 14 = Aricari farm/BR156 road, km147, municipality of Tartarugalzinho, 00º56' N; 51º14' W.

Figure 2 :

 2 Figure 2: Sonogram of the second harmonic of the echolocation call emitted byCommon Mustached Bats in French Guiana. The sonogram was produced using a 512point FFT (Fast Fourier Transform) created with R 24 (R Development Core Team, 2011) using the package Seewave[START_REF] Sueur | Rapid Acoustic Survey for Biodiversity Appraisal[END_REF]. The Frequency of Maximal Energy (FME) is measured at around 60 kHz (left) or 54 kHz (right) and corresponds to the 59 kHz and 53 kHz phonic types, respectively.

Figure 3 :

 3 Figure 3: Histograms of constant frequency (CF) values emitted by 257 Common Mustached Bats caught in French Guiana. The median constant frequency value (CF) emitted by 130 individuals of the first phonic type (left) was 53.5 kHz (standard deviation 0.62 kHz), and that of the 127 individuals of the second phonic type (right) was 59.3 kHz (standard deviation 0.68 kHz).

Figure 4 :

 4 Figure 4: Parsimony haplotype networks for 14 CO1 sequences of Common Mustached Bats corresponding to the 53 kHz (below) and 59 kHz (above) phonic types, respectively. All bats were caught in syntopy at Grotte Mathilde, French Guiana. Ovals indicate haplotypes found in only one specimen, whereas the rectangles represent haplotypes shared by 6 (59 kHz phonic type) and 5 (53 kHz phonic type) animals, respectively. Numbers in the symbols correspond to the last three digits of the vouchered specimens (MHNG-1978.xxx).

Figure 5 :

 5 Figure 5: Maximum likelihood tree showing the phylogenetic relationships among 26 CO1 barcodes of Common Mustached Bats from Amapa, Mato Grosso, French Guiana, Guyana, Suriname, Venezuela, El Salvador, Mexico, and Jamaica. Bootstrap support from ML and posterior probabilities (PP) from a Bayesian analysis are shown above and below major nodes, respectively. Labels for continental CMB are composed of the same species names as in Clare et al. (2013) followed by specimen reference (see Appendix 1

Figure 6 :

 6 Figure 6: Maximum likelihood tree showing the phylogenetic relationships among 31 Cyt b sequences of Pteronotus specimens from Central, South America and the Caribbean region. The legend is the same as for Fig. 5 except that the tree was rooted

Figure 7 :

 7 Figure 7: Distribution of forearm lengths (in mm) of the 53 kHz phonic type (white bars) and 59 kHz phonic type (black bars) Common Mustached Bats in French Guiana.Sample sizes are 111 and 89 individuals for each phonic type, respectively. Sexes and localities were not differentiated.

Figure 8 :

 8 Figure 8: Results of the stepwise discriminant function analysis [F (df 3, 18) = 64.42, p < 0.0001] including the three most discriminant cranial variables (MDT = mandibular toothrow length, BRD = braincase depth, and CCL= condylo-canine length) in 8 specimens of the 53 kHz (white bars) and 14 of 59 kHz phonic type (black bars), as well as the specimen MZUSP-35152 from Mato Grosso (grey bar).

Acknowledgements

We wish to thank and give credit to Alexandre Haquart who warned us about the existence of two classes of Common Moustached Bats emitting ultrasound calls at different constant frequencies in Saut-Pararé, French Guinana. We wish to thank also Burton K. Lim (Royal Ontario Museum, Canada) who generously provided ethanolpreserved tissues from Jamaican-caught P. parnellii and Sylvie Jouard who kindly made available a series of cleaned skulls from the personal collection of P. Charles-Dominique (MNHN France). The genetic work was supported by LIVH laboratory of Institut Pasteur de la Guyane, and by the project JAGUARS of Kwata NGO. Part of the work has benefited from an "Investissement d'Avenir" grant managed by the French Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-25-01).

Literature Cited

two phonic type CMB caught in sympatry in French Guiana. Values are expressed in kHz and correspond to the frequency containing maximum energy (FME), with mean, standard deviation (SD), minimum (min) and maximum (max) values; N is the number of bats recorded.

Locality