Laure Desutter-Grandcolas
email: laure.desutter-grandcolas@mnhn.fr

Sylvain Hugel

André Nel

Ben H Warren

Pedro Souza-Dias

Ioana C Chintauan-Marquier

Keywords: Systematics, Diagnosis, Calibration, Fossil record, Crickets

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

Crickets (Orthoptera, Gryllidea) are a major model group in biology, especially for acoustic communication and behavioral ecology [START_REF] Choe | The evolution of mating systems in Insects and arachnids[END_REF][START_REF] Gerhardt | Acoustic communication in insects and anurans[END_REF].

Evolutionary hypotheses need to be tested based on accurate phylogenies. But in the case of the crickets, few extensive phylogenetic studies are available, and only at low taxonomic levels, e.g. the subfamily Eneopterinae (Gryllidae: Robillard and Desutter-Grandcolas, 2011; Vicente et al., 2017 and references therein), Chinese species of subfamily Trigonidiinae (He et al. 2020) or Nemobiinae (Ding et al. 2020), and the genera Nudilla Gorochov, 1988(Trigonidiidae, Trigonidiinae: Mendelson and Shaw 2005, under Laupala Otte, 1994) and

Eidmanacris Chopard, 1956(Phalangopsidae: Campos et al., 2017, 2021). Using approximatively 200 terminals, [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF] proposed the first extensive molecular phylogenetic hypothesis for the infraorder Gryllidea, supporting the superfamilies Gryllotalpoidea and Grylloidea. Within Grylloidea, four families were found monophyletic, viz. the Mogoplistidae, Phalangopsidae, Gryllidae and Trigonidiidae, the latter being subdivided into the two subfamilies Nemobiinae and Trigonidiinae (not counting the subfamily Pteroplistinae considered incertae sedis within Grylloidea; [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF].

The Trigonidiidae is highly diversified worldwide (except in desert areas). Although wellseparated among crickets (it is actually recovered as the sister group of Phalangopsidae + Gryllidae + Pteroplistinae), and easily recognized 'at first glance', the Trigonidiidae and its subfamilies suffer from being poorly diagnosed, with few recognized synapomorphies that would support unambiguously the potential attributions of fossils to these clades. Yet because of the growing use of fossils to calibrate relaxed clocks in molecular phylogenetic studies [START_REF] Dos Reis | Bayesian molecular clock dating of species divergences in the genomics era[END_REF][START_REF] Wright | A systematist's guide to estimating Bayesian phylogenies from morphological data[END_REF], it has become crucial to have clear morphological character sets to clarify the positions of fossil taxa in the lineage history in relation to extant species.

Although this is true for all uses of fossils in calibrating phylogenies, it is particularly important for those methods that go beyond node dating to make explicit use of morphological characters to place ancestral forms in lineage history (e.g. fossilized birthdeath; [START_REF] Heath | The fossilised birth-death process for coherent calibration of divergence-time estimates[END_REF]).

Here we propose updated extended diagnoses for the cricket family Trigonidiidae and the subfamilies Trigonidiinae and Nemobiinae. Then we review the fossils potentially attributable to these clades. The oldest representative of the family, i.e. the recently described mid-Cretaceous Burmese amber Birmaninemobius hirsutus [START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF], is currently attributed to the Nemobiinae, but it exhibits several characters that challenge this proposal. We find evidence that it could rather belong to the stem group of the Trigonidiinae, with which it shares several synapomorphies.

Material and methods

Description of male genitalia follows Desutter (1987,1988), modified in [START_REF] Desutter-Grandcolas | Phylogeny and the evolution of acoustic communication in extant Ensifera (Insecta, Orthoptera)[END_REF]. On hind tibiae (TIII), we separate spurs (movable) from spines and serrulation (not movable); we also distinguish apical spurs from subapical spurs, and we count subapical spurs from tibial apex upward [START_REF] Hugel | Chapitre 16 Ordre des Orthoptera (Orthoptères) & sous-ordre des Orthoptera Ensifera (Orthoptères Ensifères)[END_REF]. Both apical and subapical spurs are named after their location on TIII sides, i.e. inner vs outer; apical spurs are also named after their relative position, i.e. ventral, median or dorsal. Wing venation is named after [START_REF] Desutter-Grandcolas | 3-D imaging reveals four extraordinary cases of convergent evolution of acoustic communication in crickets and allies (Insecta)[END_REF], modified after [START_REF] Schubnel | To be or not to be: postcubital vein in insects revealed by microtomography[END_REF], using observations and homology settings in X-ray tomography: these studies showed that a postcubital vein (PCu thereafter) is present in nearly all neopteran insects, including Orthoptera; in Grylloidea, tomographic observations showed that the stridulatory file is located on this PCu vein, and neither on the posterior cubital vein or first anal vein as proposed by earlier authors (see [START_REF] Desutter-Grandcolas | Phylogeny and the evolution of acoustic communication in extant Ensifera (Insecta, Orthoptera)[END_REF].

For taxonomy we follow [START_REF] Cigliano | Orthoptera Species File[END_REF], with the family-rank classification from the clades supported in [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF]. An identification key for cricket families and trigonidiid subfamilies is given in Table 2. The characters of the family and subfamilies have been checked using the specimens deposited in the collections of the Trigonidiidae [START_REF] Bruner | South American crickets, Gryllotalpoidea and Achetoidea[END_REF]: 401 -Chopard 1949: 671 -Chopard 1969: 273 -Desutter 1987: 224 -Desutter 1988: 344, 364.

Trigonidiinae [START_REF] Kirby | A synonymic catalogue of Orthoptera. Vol. 2. Orthoptera Saltatoria. Part 1. Achetidae and Phasgonuridae[END_REF]: 77 -Chopard 1943: 241 -Chopard 1968: 303.

Phylogeny. Fig. [START_REF] Saussure | Recherches scientifiques pour servir à l'histoire de la faune de l'Amérique centrale et du Mexique. VIème partie[END_REF][START_REF] Saussure | Mélanges orthoptérologiques. VIème fascicule. Gryllides (2 ème partie)[END_REF]Saussure, , 1893;; for example), later recognized as a subfamily (e.g., [START_REF] Beier | Handbuch der Zoologie. IV. Band: Arthropoda -2. Hälfte: Insecta. 2. Teil: Spezielles. 9. Saltatoria[END_REF][START_REF] Otte | Crickets from Australia (Orthoptera: Gryllidae)[END_REF][START_REF] Scudder | Guide to the genera and classification of the North American Orthoptera found north of Mexico[END_REF], or a family (e.g., [START_REF] Bruner | South American crickets, Gryllotalpoidea and Achetoidea[END_REF][START_REF] Brunner Von Wattenwyl | Système des Gryllides[END_REF][START_REF] Chopard | Ordre des Orthoptères[END_REF][START_REF] Chopard | The fauna of India and adjacent countries. Orthoptera[END_REF][START_REF] Vickery | A monograph of Orthopteroid insectes[END_REF].

By contrast, Nemobiinae have long been gathered with field crickets, as a subfamily or a tribe, i.e. within the Gryllidae / Gryllinae (e.g., [START_REF] Brunner Von Wattenwyl | Système des Gryllides[END_REF][START_REF] Brunner Von Wattenvyl | Prodromus der Europaïschen Orthopteren[END_REF][START_REF] Chopard | Ordre des Orthoptères[END_REF][START_REF] Chopard | Pars 10: Gryllides: Fam. Gryllidae: Subfam. Gryllinae (Trib. Gymnogryllini, Gryllini, Gryllomorphini, Nemobiini)[END_REF][START_REF] Saussure | Recherches scientifiques pour servir à l'histoire de la faune de l'Amérique centrale et du Mexique. VIème partie[END_REF][START_REF] Saussure | Mélanges orthoptérologiques. VIème fascicule. Gryllides (1ère partie)[END_REF]Saussure, , 1897;;[START_REF] Vickery | A monograph of Orthopteroid insectes[END_REF]. This hypothesis has recently been invalidated by morphological observations and molecular phylogenies (see infra), but as early as 1930, Shiraki (1930) considered both groups as equal rank subfamilies within a Gryllidae family that comprised all crickets (= Gryllidea infraorder), a classification followed by several authors, perhaps because of the lack of a clear phylogenetic system of classification for crickets (e.g., Otte, 1994;[START_REF] Otte | Crickets from Australia (Orthoptera: Gryllidae)[END_REF][START_REF] Otte | Carribean crickets. The Orthopterists[END_REF][START_REF] Rentz | Grasshopper country: the abundant orthopteroid insects of Australia[END_REF].

Using morphological characters, and especially strong similarities in male genitalic structures, the close relationship between Trigonidiinae and Nemobiinae has been documented by [START_REF] Gorochov | System and morphological evolution of crickets from the family Gryllidae (Orthoptera) with description of new taxa[END_REF] and [START_REF] Desutter | Structure et évolution du complexe phallique des Gryllidea (Orthoptera) et classification des Grylloidea néotropicaux. 1ère partie[END_REF][START_REF] Desutter | Structure et évolution du complexe phallique des Gryllidea (Orthoptères) et classification des genres néotropicaux de Grylloideadeuxième partie[END_REF]. [START_REF] Desutter | Structure et évolution du complexe phallique des Gryllidea (Orthoptera) et classification des Grylloidea néotropicaux. 1ère partie[END_REF][START_REF] Desutter | Structure et évolution du complexe phallique des Gryllidea (Orthoptères) et classification des genres néotropicaux de Grylloideadeuxième partie[END_REF] proposed to group Trigonidiinae and Nemobiinae subfamilies in one family within the Grylloidea superfamily, while [START_REF] Gorochov | System and morphological evolution of crickets from the family Gryllidae (Orthoptera) with description of new taxa[END_REF][START_REF] Gorochov | System and evolution of the suborder Ensifera (Orthoptera). Parts 1, 2[END_REF] Diagnosis. In addition to the characters of the family. Body very thin. Head small, triangular in front view (Fig. 1A andB); scapes longer than wide. Eyes most often protruding in dorsal view, much separated from fastigium by antennal insertions. Lateral ocelli most often not distinct. Pronotum narrower than head in dorsal view (Fig. 1A andB). Claws serrated (Fig. 1C), not only bifid. Second tarsomeres wide and flat (Fig. 1D). Fore tibiae with only one apical spur, medio-ventral in location (Fig. 1E andF). Hind tibiae and hind femora almost equal in length. Hind femora thin; ventral gutter not laterally widened on femur inner side.

Hind tibiae thin; with three inner and three outer subapical spurs; with two long, inner and usually three (rarely two) shorter, outer apical spurs, the median outer spur longer than ventral and dorsal outer spurs. Subapical spurs of hind tibiae usually short compared to hind basitarsomere, and subequal in length on each side (Fig. 1G); when longer, spurs about equal in size, not increasing in length along tibia length as in Nemobiinae. Cerci most often short, distinctly wider and convergent at anterior half (Fig. 2C). Males: forewing condition variable, some species with corneous forewings with longitudinal venation only, identical in males and females (Fig. 2E-G); when present, stridulum with a well-developed mirror, not crossed by a transverse vein but often with a rounded false vein, and usually clearly separated from apical cells (Fig. 2H); apical field strongly reduced. Male genitalia (Fig. 3) with well-developed pseudepiphallic parameres; pseudepiphallic lophi dorsal in position; ectophallic arc almost always present; endophallic sclerite most often well-developed. Females: wing condition often similar to that of males, except for the stridulum. Ovipositor flattened laterally and distinctly upcurved; apex distinctly longer (often more than one third of ovipositor length) and larger than in Nemobiinae (compare Fig. 2A and B and Fig. 6A andB).

Inner classification. The Trigonidiinae are currently divided into two tribes, the world-wide Trigonidiini Saussure, 1874 and the New World Phylloscyrtini Chopard, 1968, characterized by the foliaceous shape of the last article of maxillary palpi; 16 genera are currently not classified in a tribe [START_REF] Cigliano | Orthoptera Species File[END_REF]. The molecular data published by [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF] could not test this subdivision of the subfamily, as the Phylloscyrtini were represented by only one species, but this terminal was found nested among the other trigonidiine species. The monophyly of the two tribes will consequently have to be tested further.

Included genera.

Owing to the morphological distinctiveness of the Trigonidiinae, all the genera classified within the subfamily certainly belong to it [START_REF] Cigliano | Orthoptera Species File[END_REF]. The monophyly of the genera is however far from ascertained, as shown by molecular results for Anaxipha [START_REF] Saussure | Recherches scientifiques pour servir à l'histoire de la faune de l'Amérique centrale et du Mexique. VIème partie[END_REF][START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF]. Recent taxonomic studies insist on the necessity to reconsider generic definitions (e.g., Hugel 2012 for Metioche Stål, 1877;

Walker & Funk 2014 for Anaxipha; [START_REF] Tan | A taxonomic study on the Bornean and Philippines sword-tailed crickets in the genus Rhicnogryllus Chopard, 1925 (Orthoptera: Trgonidiidae (sic); Trigonidiinae)[END_REF]for Rhicnogryllus Chopard, 1925;Gorochov et al. 2018).

Habitat. Contrary to Nemobiinae (see infra), Trigonidiinae are found in a narrow range of habitats (Fig. 4). They live most often on plants, in both open and forested areas, even in disturbed environments. In forest, some species can be found in the leaf litter, hiding during the day and foraging at night on small plants; other species live in understorey shrubs and young trees, or in the canopy. Species with long hindtibial spurs are known to move on water surface, as Hydropedeticus vitiensis Miall andGilson, 1902 (Chopard 1938). Some species exhibit very bright colours, such as the light blue Rhicnogryllus lepidus Chopard, 1962 from Tanzania [START_REF] Desutter-Grandcolas | Rhicnogryllus lepidus Chopard, 1961, un Trigonidiinae bleu de Tanzanie (Grylloidea, Trigonidiidae)[END_REF], the blue and orange Metioche (Superstes) superbus Walker & Funk 2014). However, despise their small size, their calls are powerful: in French Guianese rainforests, for example, trigonidiine calls are one of the dominant calls in the diurnal soundscape (J. Anso & LDG, pers. obs.). These loud emissions could be related to the structures of their stridulum, as hypothesized by [START_REF] Desutter-Grandcolas | Chant et appareil stridulatoire de deux Trigonidiinae originaires d'Equateur (Orthoptera: Grylloidea: Trigonidiidae)[END_REF]. The power of trigonidiine calls can be increased by strategies for sound amplification, as the baffling, a behaviour typical of Oecanthinae crickets [START_REF] Forest | Power output and efficiency of sound production by crickets[END_REF][START_REF] Prozelsky-Schulze | Use of a self-made sound baffle by a tree cricket[END_REF]), but also observed in Trigonidiinae. A good example is Natula sp. in the Guadeloupe island, which sing on Ipomea leaves (Convolvulaceae) (SH & LDG pers. obs., Fig. 4C).

Distribution. World wide.

Subfamily Nemobiinae Saussure, 1877

Nemobiites Saussure, 1877: 65 -Chopard 1949: 668.

Gryllinae [START_REF] Kirby | A synonymic catalogue of Orthoptera. Vol. 2. Orthoptera Saltatoria. Part 1. Achetidae and Phasgonuridae[END_REF]: 13. Nemobiidae Bruner 1916: 368 -Liu et al. 1998: 55. Nemobiinae Shiraki 1930: 186, 195 -Chopard 1943: 238 -Chopard 1969: 156. Nemobiini Chopard 1967: 158. Diagnosis. In addition to the characters of the family: body thicker and wider than in Trigonidiinae. Head wider, variable in shape. Eyes most often not protruding dorsally. Lateral ocelli distinct. Pronotum broader than in Trigonidiinae and bearing longer, stronger setae (Fig. 5A andB). Claws regular, not serrated. Second tarsomeres neither widened, nor flat (Fig. 5C). Fore tibiae with two apical, ventral spurs (Fig. 5D andE). Hind femora usually wider than in Trigonidiinae and much longer than hind tibiae; ventral gutter wide, largely extended laterally on inner side of hind femur, the hind tibiae being encased at rest in the gutter. Hind tibia subapical spurs longer than in Trigonidiinae, remarkably long in some species (Fig. 5F andG); proximal subapical spurs often much shorter than distal subapical spurs, especially in species with elongated spurs (Fig. 5F); with three outer apical spurs, and two or three inner apical spurs. Cerci often longer than in Trigonidiinae, and straight (Fig. 6C and D). Males: hind tibia subapical spurs sometimes glandular (Fig. 5G, fourth inner subapical spur in Pteronemobius obscurior Chopard, 1957). Forewing condition variable, from fully developed to absent, from thin to coriaceous; when present, stridulum with a reduced mirror more or less distinct from other cells of fore wing apical field (Fig. 6E andF),

Phylogeny.

or lacking, as in some species of Hygronemobius Hebard, 1913. Male genitalia (Fig. 7) with reduced pseudepiphallic parameres, and without a proper ectophallic arc, the sclerotization of ectophallic apodemes prolonged at base of ectophallic fold. Female: fore wing condition variable. Ovipositor not flattened laterally, and straight (Fig. 6A andB Hemigryllus has long been classified within the Nemobiinae [START_REF] Bruner | South American crickets, Gryllotalpoidea and Achetoidea[END_REF][START_REF] Chopard | Pars 10: Gryllides: Fam. Gryllidae: Subfam. Gryllinae (Trib. Gymnogryllini, Gryllini, Gryllomorphini, Nemobiini)[END_REF][START_REF] Kirby | A synonymic catalogue of Orthoptera. Vol. 2. Orthoptera Saltatoria. Part 1. Achetidae and Phasgonuridae[END_REF]Otte, 1994;[START_REF] Saussure | Mélanges orthoptérologiques. VIème fascicule. Gryllides (1ère partie)[END_REF]etc.), but morphological characters clearly demonstrate that it does not belong to this clade [START_REF] Desutter | Structure et évolution du complexe phallique des Gryllidea (Orthoptera) et classification des Grylloidea néotropicaux. 1ère partie[END_REF][START_REF] Desutter | Structure et évolution du complexe phallique des Gryllidea (Orthoptères) et classification des genres néotropicaux de Grylloideadeuxième partie[END_REF][START_REF] Desutter | Etude phylogénétique, biogéographique et écologique des Grylloidea néotropicaux (Insectes, Orthoptères)[END_REF][START_REF] Gorochov | System and morphological evolution of crickets from the family Gryllidae (Orthoptera) with description of new taxa[END_REF].

According to the molecular topology of Chintauan-Marquier et al. (2016, clade G), Hemigryllus is a Gryllidae s. str., not a Trigonidiidae, but its position within the family is still uncertain.

As mentioned above, we transfer here Lissotrachelus Bruner von Wattenwyl, 1893 to the Nemobiinae. This genus was previously considered a Pentacentrinae (Gryllidae) by [START_REF] Chopard | The fauna of India and adjacent countries. Orthoptera[END_REF]. More recently Lissotrachelus (Lissotrachelinae) was considered Trigonidiidae (Yin and Liu 1995), and according to the shape of second tarsomeres, He and Chen (2020) suggested that Lissotrachelus may belong to Nemobiinae, a hypothesis further supported by the shape of the head, the armature of the hind tibiae, the male pseudephiphallus and the female ovipositor.

Two other genera are presently classified in the Lissotrachelini [START_REF] Cigliano | Orthoptera Species File[END_REF]), i.e. Trigonidomimus Caudell, 1912and Tohila Hubbell, 1938. These genera are here transferred in Pentacentrinae incertae sedis; their classification will be studied further elsewhere (Hugel in prep.).

Habitat. Most Nemobiinae live and forage in the leaf litter (Fig. 8A [START_REF] Chopard | Orthoptères fossiles et subfossiles de l'ambre et du copal[END_REF]; and Anaxipha dominica Vickery & Poinar, 1994 (20.43-13.65 My, Miocene Dominican Amber). Liaonemobius tanae Ren, 1998 (125.45-122.46 Abanaxipha longispina is based on a nearly complete female (specimen LEM n°50, amber inclusion). [START_REF] Gorochov | New and little known orthopteroid insects (Polyneoptera) from fossil resins: Communication 3[END_REF] later attributed to this species a complete male (NMNH no. 503374, Acc. 371428, Woodruff (collection reg.) 8820) and an incomplete female (NMNH no. 502562, Acc. 371428, Woodruff (collection reg.) 5329). The female type has a long and straight ovipositor, and its hind tibial subapical spurs are very long, greatly increasing toward tibial apex, being apparently very different from those of the male (compare Fig. 8f and Fig. 8g in [START_REF] Gorochov | New and little known orthopteroid insects (Polyneoptera) from fossil resins: Communication 3[END_REF]. The male fossil (Fig. 9A) is a Trigonidiinae by its small, triangular head with large protruding eyes, scape shape, narrow pronotum, flattened second tarsomeres, fore wing harp vein, absence of serrulation on hind tibiae and hind basitarsomeres, and equal-sized subapical spurs. The systematic placement of the female type of A. longispina will have to be checked however by reexamination of the fossil (which could not be done for the present study).

Abanaxipha incongrua (described on posterior part of a female, specimen LEM no.

54, amber inclusion) differs from A. longispina by its short tibial spurs and shorter ovipositor.

Cyrtoxipha (?) electrina (complete female, specimen NMNH no. 504374, Acc.

371428, Woodruff (collection reg.) 8820, Brodzinsky/Lopez-Penha Collection, amber inclusion) presents trigonidiine fore and hind legs, and ovipositor; its fore tibia could have only one apical spur (Fig. 8h in Gorochov 2010) but this should be checked by reexamining the fossil. Cyrtoxipha (?) illegibilis (incomplete remains of a male, specimen NMNH no.

506712, Acc. D, Woodruff (collection reg.) 11585, Brodzinsky/Lopez-Penha Collection, amber inclusion) resembles the previous species by its fore leg.

Rhicnogryllus zeuneri (BMNH I. 13768) and Amusurgus africanus (BMNH, Geol.

Dept. no. 58584, 17680; Fig. 9B) belong to their respective modern trigonidiine genera [START_REF] Chopard | Orthoptères fossiles et subfossiles de l'ambre et du copal[END_REF]: note that the drawing of left hind tibia incorrectly shows a full serrulation which is lacking on right tibia). Anaxipha dominica, described on the basis of three females (specimens CAL no. 1, CAL no. 8, LEM no. 56, amber inclusions), is clearly a Trigonidiinae, but its generic attribution is questionable, as generic diagnosis relies on male characters.

Baltonemobius fossilis (juvenile female, specimen ZIN Balt. 3, amber inclusion; Fig. 9C) belongs to Trigonidiidae according to the lack of spines on its hind tibia and basitarsomere, and the shape and thick setation of its head and pronotum; its scape is typical of Nemobiinae.

Pteronemobius (?) anglicus has been described by [START_REF] Zeuner | Descriptions of new genera and species of fossil Saltatoria (Orthoptera)[END_REF] based on an incomplete female forewing (specimen NHMUK In. 24778, Hooley collection). Its attribution to the Nemobiinae is questionable. Gorochov (2019: 326) indicated that it is based on a 'poorly preserved imprint that may be determined only as belonging to Gryllidae' (= Grylloidea in the taxonomic system adopted here).

The very young juvenile cricket described by [START_REF] Chopard | Orthoptères fossiles et subfossiles de l'ambre et du copal[END_REF] as a Nemobius sp. from the Eocene (Coll. Kühl, Kasten 31, Geologisches Institut, Berlin) is actually a Nemobiinae, but its generic attribution is questionable.

Most of the fossils listed above are informative enough to be easily attributed to one subfamily or the other among Trigonidiidae. But the placements of Mesozoic specimens are more problematic. The Early Cretaceous Liaonemobius tanae Ren, 1998 (described after male specimens LB97301 and 97302, part and counterpart, and specimen LB97303, NGMC) was originally described as a Trigonidiinae [START_REF] Ren | First record of fossil crickets (Orthoptera: Trigonidiidae) from China[END_REF][START_REF] Meng | Current knowledge of research on Orthoptera fossils in China[END_REF]), and it is currently placed in the Gryllidae [START_REF] Cigliano | Orthoptera Species File[END_REF]). Nevertheless, this fossil (Fig. 9D) is clearly not a cricket, as shown by the lack of club-shaped setae on the inner side of the cercus, which represent one of the synapomorphies of Gryllidea [START_REF] Desutter-Grandcolas | Phylogeny and the evolution of acoustic communication in extant Ensifera (Insecta, Orthoptera)[END_REF]. Liaonemobius tanae can be considered a young nymph of an Elcanidae because it bears the elongated leaflike spurs on hind tibia typical of this family (Fig. 3 The position of Birmaninemobius within Trigonidiidae is a very realistic hypothesis, on the basis of several characters of the family, some but not all of which were considered by [START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF], namely the small triangular head (Fig. 9F), the lack of serrulation on hind tibia (Fig. 9G) and hind basitarsus (Fig. 9H) and the very large fastigium (Fig. 2F). It also has several large bristles on the body and a much reduced apical field on the forewing (Fig. 9I).

The harp of the male stridulatory apparatus is crossed by two veins (Fig. 9I), one oblique and longitudinal, typical of Trigonidiidae, and one more proximal and transverse.

According to [START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF] Hygronemobius Hebard, 1913 [START_REF] Desutter-Grandcolas | New Nemobiinae crickets from Guianese and Peruvian Amazonia (Orthoptera, Grylloidea, Trigonidiidae)[END_REF] or the fossil Baltonemobius fossilis. Finally Birmaninemobius stridulum has a distinct mirror (plesiomorphy), which is not clearly separated from other fore wing distal cells (Fig. 1A in [START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF].

To summarize, Birmaninemobius has several apomorphies of the Trigonidiinae, as listed below:

 scape longer than wide (Fig. 9F) (short, and wider than long in Nemobiinae);

 fore tibia bearing only one long apical spur, large and medio-ventral in location (Fig. Birmaninemobius lacks, however, some of the autapomorphies of the Trigonidiinae, such as the serrated claws (simple in B. hirsutus: Fig. 9H), or the rounded false vein observed in the male mirror of many (but not all) extant Trigonidiinae (subdivided into three cells in B.

hirsutus: Fig. 9I). Its cerci are long, thin and straight (Fig. 9E), a plesiomorphic trait in crickets. Finally, Xu et al. (2020: Fig. 3F) draw the ventral gutter of the hind femur straight, not widened on the femur inner side, but this feature is not clear on the photo of the fossil (Fig. 9G).

Birmaninemobius has also unique features, such as the elongate tarsi with three long tarsomeres (Fig. 9E), or the shape and venation of the mirror (Fig. 9I).

Because of the mosaic of characters described, Birmaninemobius hirsutus could better be classified in the stem group of the Trigonidiinae, and not as a member of the Nemobiinae.

Discussion

Phylogenetic and evolutionary studies increasingly rely on fossils to date cladogenetic events, most often reconstructed using molecular data [START_REF] Ronquist | A total-evidence approach to dating with fossils, applied to the early radiation of the Hymenoptera[END_REF] for Hymenoptera; Studies that consider both extant and fossil taxa can also relate the evolution of the characters of interest to the palaeoecology of the corresponding taxa, to past environments, or more generally to past events. In addition to dating evolutionary events in the Deep Past, such studies can also reconcile the taxonomy of extant and fossil taxa to produce a unified taxonomy that is consistent throughout the relevant stretch of lineage history, including both living and ancestral forms. All these studies need however that fossils are attributed to the clades represented in the phylogeny. In the case of crickets, this is hindered by two main issues, i.e. the lack of a reference classification derived from accurate phylogenetic hypotheses, and the lack of morphological synapomorphies to define the different clades, including those derived from phylogenetic topology.

Cricket classification

Since the beginning of the 20th century, crickets have been arranged in two main classificatory systems, considering all true crickets either as one family, the Gryllidae s. l., or as the superfamily Grylloidea with up to 12 or 13 families, including the Gryllidae s. str. [START_REF] Bruner | South American crickets, Gryllotalpoidea and Achetoidea[END_REF][START_REF] Chopard | Ordre des Orthoptères[END_REF][START_REF] Vickery | Taxon ranking in Grylloidea and Gryllotalpoidea[END_REF]McE Kevan 1982;[START_REF] Desutter | Structure et évolution du complexe phallique des Gryllidea (Orthoptères) et classification des genres néotropicaux de Grylloideadeuxième partie[END_REF]). Many fossils are / have been described as 'Gryllidae' without further classificatory precision, which limits greatly their use as calibration points in molecular phylogenetic studies.

Xu et al. ( 2020) use a different classificatory system, never used before for crickets.

They considered four families within Grylloidea, i.e. the Gryllotalpidae, Mogoplistidae, Trigonidiidae, and Gryllidae, which means either that they include in their 'Gryllidae' all the Grylloidea that are the sister group of Trigonidiidae (without giving a new diagnosis of this clade), or that they ignored the Phalangopsidae and the Pteroplistinae, which represent a large diversification within Grylloidea [START_REF] Cigliano | Orthoptera Species File[END_REF]. Either way, this option cannot but generate confusion in cricket taxonomy.

The molecular topology of [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF], based on the study of more than 200 terminals, proposed a backbone for a phylogenetic classification of crickets:

based on their molecular tree topology, these authors proposed to separate the whole cricket clade (infra-order Gryllidea) into two superfamilies (Gryllotalpoidea and Grylloidea), and they recognize within Grylloidea four clades to which they assign a familial rank, the Mogoplistidae, Trigonidiidae, Phalangopsidae, and Gryllidae, with the Pteroplistinae incertae sedis within Grylloidea. This classification proposal must be completed by clear diagnoses of monophyletic familial and subfamilial clades, and clear morphological synapomorphies, to stabilize the cricket classification system. In this paper, we give clear diagnoses of the Trigonidiidae and its two subfamilies Trigonidiinae and Nemobiinae, allowing us in turn to reconsider the attribution of the fossils placed in these clades. We also propose a key to identify extant cricket families, using morphological characters (Table 2).

Trigonidiidae (TD), Trigonidiinae (TN) and Nemobiinae (NE) can be (and are currently) identified by unique combinations of morphological characters, listed explicitly in the diagnoses given above and called here TD0, TN0 and NE0 respectively: these combinations of characters are a mix of homoplastic characters (i.e. size, setation, presence / absence of more or less labile characters, etc.), which, even though they allow easy recognition of these taxa, i.e."at first glance", are not a substitute for true apomorphies. More (NE3) the shape of hindlegs, with a wide femur having a large gutter on ventro-lateral margin, and shorter tibia; (NE4) shape of ovipositor (including apex).

Cricket fossils

Many fossil crickets are known by fore wing imprints showing a cricket-like stridulatory apparatus, viz. with a file, harp and a mirror (but a file, harp and mirror are also present in the Hagloidea). Grylloidea have a file on the vein PCu while their vein CuPb is reduced to a reduced intercalary vein between CuPa and PCu, as main apomorphy in the stridulatory apparatus [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF][START_REF] Schubnel | To be or not to be: postcubital vein in insects revealed by microtomography[END_REF]. Fossils

showing these structures can be accurately attributed to this clade, even without further precision; however their relationships with the different families are problematic, because only few cricket clades are characterized by apomorphic changes on their forewing venation, among other morphological characters. This is the case for example of the Trigonidiidae (see above, Fig. 2H, and Fig. 5E andF), of the extant subfamily Phaloriinae (Phalangopsidae) and the fossil family Baissogryllidae, which is currently considered the sister group of the rest of the 'Grylloidea' without extended phylogenetic evidence [START_REF] Heads | On the placement of the Cretaceous orthopteran Brauckmannia groeningae from Brazil, with notes on the relationships of Schizodactylidae (Orthpotera, Ensifera)[END_REF]. The recent discoveries of complete and ancient fossils in amber are great opportunities to improve knowledge of fossil crickets and thereby improve evolutionary studies of this clade.

Based on current knowledge, the only fossils that can be attributed to Nemobiinae are Baltonemobius fossilis (TD1, TD2, NE3) and Nemobius sp. (NE0, NE1?), two juveniles which would be difficult to include in a morphological phylogeny owing to available characters. They can be used to trace back the existence of the subfamily to Middle -Late Eocene at most.

More fossils can be attributed to the Trigonidiinae (see Table 1), but most of them are rather recent, the oldest being Miocene in age. Moreover, several trigonidiine fossils are incomplete remains and most are females, which limit their potential use in morphological phylogenies. Males are known for ?Cyrtoxipha illegibilis (incomplete fossil, Miocene) and

Rhycnogryllus zeuneri (after Chopard 1936: TD0, TD1, TD2, TN0; claws described as not serrated by [START_REF] Chopard | Orthoptères fossiles et subfossiles de l'ambre et du copal[END_REF], but extant species of the genus have their claws very weakly serrated: this character will have to be checked on the fossil), not mentioning the male attributed to Abanaxipha longispina (TD0, TD1, TD2, TD3, TN0; TN1 and TN2 to be checked on fossil; Gorochov 2010), which will have to be compared to the female type of that species. Female fossils of Trigonidiinae are Amusurgus africanus (TD0, TN0, TN5, TN6; TD1, TD2, TN1 and TN2 to be checked on fossil), ?Cyrtoxipha electrina (TD0, TD1, TD2, TN0, TN1, TN5, TN6; TN2 to be checked on fossil), Anaxipha dominica (TD0, TD1, TD2, TN0, TN5, TN6), and Abanaxipha incongrua (incomplete fossil, Miocene).

There have been in the past much confusion between the Trigonidiinae and the Pentacentrinae (Gryllidae), as both could be described as 'small, elongate and thin crickets with long wings', in the lack of precise apomorphies of each group (see [START_REF] Gorochov | New and little known orthopteroid insects (Polyneoptera) from fossil resins: Communication 3[END_REF]. The list of above apomorphies of Trigonidiinae should help avoiding such errors.

Birmaninemobius hirsutus is the oldest fossil clearly belonging to the Trigonidiidae on the base of several apomorphies (TD0, TD1, TD2, TN1; TN3 to be checked on the fossil). It originates from the deposits of Hkamti site (Hkamti District, Sagaing Region, Myanmar), about 80 km southwest of the Angbamo site. Radiometric data established an early Cenomanian age (98.79±0.62 My) for Kachin amber, based on zircons from volcanic clasts found within the amber-bearing sediments [START_REF] Shi | Age constraint on Burmese amber based on UePb dating of zircons[END_REF]. Some ammonites found in the amber-bearing deposits corroborate a late Albian-early Cenomanian age [START_REF] Cruickshank | Geology of an amber locality in the Hukawng Valley, Northern Myanmar[END_REF][START_REF] Yu | An ammonite trapped in Burmese amber[END_REF]. Owing to its morphological characters, B. hirsutus is here attributed to the stem group of the Trigonidiinae, not to the Nemobiinae crown group (contra [START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF]. This allows the divergence of these two subfamilies to be dated, which is much deeper in the past than the dates provided by the fossils classified in the crown groups of both subfamilies. Birmaninemobius hirsutus is also the oldest fossil in the evolutionary history of crickets for dating the whole Trigonidiidae family, and therefore an important reference data point for future phylogenies of the Orthoptera. Abbreviations: ec. ap., ectophallic apodeme; ec. arc, ectophallic arc; ec. f., ectophallic fold; en. ap., endophallic apodeme; en. s., endophallic sclerite; ps. a. l., pseudepiphallic apical lobe; ps. p., pseudepiphallic paramere; ps. s., pseudepiphallic sclerite; r., rami. Modified from [START_REF] Desutter-Grandcolas | Chant et appareil stridulatoire de deux Trigonidiinae originaires d'Equateur (Orthoptera: Grylloidea: Trigonidiidae)[END_REF] and [START_REF] Bland | An annotated list of Orthoptera from St Eustatius and Saba, Dutch West Indies, with descriptions of two new cricket species (Trigonidiidae, Mogoplistidae)[END_REF]. Modified from [START_REF] Desutter-Grandcolas | New Nemobiinae crickets from Guianese and Peruvian Amazonia (Orthoptera, Grylloidea, Trigonidiidae)[END_REF] and Desutter-Grandcolas (1997). Table 1. Reanalysis of the familial / subfamilial attribution of the fossils described, or attributed today [START_REF] Cigliano | Orthoptera Species File[END_REF]

Figure and table captions

  Updated diagnoses for the cricket family Trigonidiidae (Insecta: Orthoptera: Grylloidea) and its subfamilies (Trigonidiinae, Nemobiinae), with a review of the fossil record Laure Desutter-Grandcolas, Sylvain Hugel, André Nel, Ben H. Warren, Pedro Souza-Dias, Ioana Chintauan-Marquier To cite this version: Laure Desutter-Grandcolas, Sylvain Hugel, André Nel, Ben H. Warren, Pedro Souza-Dias, et al.. Updated diagnoses for the cricket family Trigonidiidae (Insecta: Orthoptera: Grylloidea) and its subfamilies (Trigonidiinae, Nemobiinae), with a review of the fossil record. Zoologischer Anzeiger, 2021, 294, pp.80-91. 10.1016/j.jcz.2021.06.004. hal-03366682 1 Introduction

Hugel, 2012

 2012 from Rodrigues island[START_REF] Hugel | Trigonidiinae crickets from Rodrigues Island: from widespread Pantropical species to critically endangered endemic species[END_REF]; Fig.4D), the bright yellow/light green Phylloscirtus amoenus Burmeister, 1880[START_REF] Martins | Recognition characters and new records of two species of Phylloscyrtini (Orthoptera, Gryllidae, Trigonidiinae) from southern Brazil. Iheringia, ser. Zoologia[END_REF], or contrasted patterns in Rhicnogryllus species[START_REF] Tan | A taxonomic study on the Bornean and Philippines sword-tailed crickets in the genus Rhicnogryllus Chopard, 1925 (Orthoptera: Trgonidiidae (sic); Trigonidiinae)[END_REF] or Homoeoxipha Saussure, 1874 for example.Acoustic communication.Owing to their small size, Trigonidiinae emit signals at relatively high frequencies for crickets, around6-8 kHz (Bennet-Clark 1998;[START_REF] Martins | Recognition characters and new records of two species of Phylloscyrtini (Orthoptera, Gryllidae, Trigonidiinae) from southern Brazil. Iheringia, ser. Zoologia[END_REF]

Fig. 3 in

 3 Chintauan-Marquier et al., 2016, clade C2. Type genus. Nemobius Serville, 1839.

  and Fig.4in[START_REF] Ren | First record of fossil crickets (Orthoptera: Trigonidiidae) from China[END_REF].The mid-Cretaceous Burmese amber Birmaninemobius hirsutus[START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF] (Fig.9E) has been described as the oldest Nemobiinae within the Trigonidiidae[START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF] male specimen NIGP172331). The characters given by the authors to exclude the taxon from Gryllotalpoidea, Gryllidae, and Mogoplistidae (the only taxonomic alternatives of[START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF]) and place it within the Trigonidiidae are 'slender prothoracic legs which are apparently not for digging, sturdy metafemora (approximatively 1.5 times as wide as long), metatibia with slender movable spines, small-sized body (shorter than 10 mm) without scales'. If fore legs not adapted for digging can show (among other characters) that Birmaninemobius does not belong to the mole crickets (Gryllotalpoidea), the lack of scales cannot exclude the possibility of Mogoplistidae (which includes not only the scale-bearing Mogoplistinae, but also the scale-devoided Malgasiinae); also the presence of movable 'spines' (an erroneous name for spurs) cannot exclude the possibility that Birmaninemobius belongs to Gryllidae, as most extant gryllid subfamilies have spurs on the hind tibia.[START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF] did not consider the possibility that Birmaninemobius could belong to the Phalangopsidae, the most diverse cricket clade with hind tibial spurs, while the lack of spurs on hind tibia is often considered a character of the Gryllinae s. str.[START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF] seem clearly confused by the burden of past non-phylogenetic taxonomy of crickets, and the numerous changes in taxonomic levels. From this point of view, the families adopted by[START_REF] Cigliano | Orthoptera Species File[END_REF] following the phylogenetic topology of[START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF], are a clear framework for cricket taxonomy, even though subfamilies and tribes are still awaiting confirmation with further phylogenetic studies[START_REF] Hugel | Chapitre 16 Ordre des Orthoptera (Orthoptères) & sous-ordre des Orthoptera Ensifera (Orthoptères Ensifères)[END_REF].

,

  Birmaninemobius cannot belong to the Trigonidiinae because it has three inner and three outer apical spurs on hind tibiae, and because its second tarsomeres are not flattened. It could belong to the Nemobiinae because it has 'prominent bristles on vertex and frons, metatibia armed with three pairs of long and movable spines on the upper margin, and the second metatarsomere compressed without adhesive pads'. But none of these characters are exclusive apomorphies of Nemobiinae or Trigonidiinae, and none can be used to justify the classification of Birmaninemobius in the crown group of one of these subfamilies: both subfamilies have prominent bristles on vertex and frons (plesiomorphy); all Trigonidiinae have three pairs of subapical spurs on hind tibia, while many Nemobiinae have more than three pairs of subapical spurs on hind tibia (homoplasic characters). The shape of the second tarsomere differs among the extant Trigonidiinae, which lives mostly on plants, and the Nemobiinae; but this feature can be found in many other cricket groups (homoplasic character) and is related to cricket ways of life[START_REF] Hugel | Chapitre 16 Ordre des Orthoptera (Orthoptères) & sous-ordre des Orthoptera Ensifera (Orthoptères Ensifères)[END_REF]. The presence of three inner and three outer apical spurs on hindtibiae is plesiomorphic in crickets, and variable in Nemobiinae: the absence of the first (ventral) inner spur is thus observed in the Trigonidiinae and in some Nemobiinae, as the genus

  9H; unnoticed in Xu et al. (2020)?) (two ventral spurs in Nemobiinae); There are three additional non conclusive characters that could group Birmaninemobius with Trigonidiinae: Xu et al. (2020) described a median ocellus, but not the lateral ocelli, which cannot be seen on their illustrations: the lack of lateral ocelli is an apomorphy of the Trigonidiinae, while their presence is plesiomorphic in crickets. Xu et al. (2020: Fig. 3E) mentioned that male genitalia of B. hirsutus have distinct pseudepiphallic parameres. The extruding structures figured in Xu et al. (2020: Fig. 3I, G, K) could rather be part of a broken spermatophore, but if the presence of pseudepiphallic parameres was confirmed, it would be an additional trigonidiine character, as Nemobiinae have no developed parameres. Finally, the eyes of B. hirsutus are protruding dorsally (Fig. 9E, F), a character observed in most Trigonidiinae and few Nemobiinae.

  Lukashevich and Ribeiro 2019, for Diptera; Drohojowska et al. 2020 for Hemiptera; King and Beck 2020 for Mammals; Zhang and Wang 2020 for Aves; Pyron 2011 for Lissamphibia).

  specifically, within the Grylloidea, the apomorphies of the Trigonidiidae are (TD1) hindtibiae rounded dorsally and not serrulated; (TD2) hind basitarsomeres not serrulated; (TD3) when present, male stridulum with a harp crossed by only one, longitudinal, oblique vein; (TD4) male genitalia very small, elongate and flat, with rami not separated from pseudepiphallic sclerite. The apomorphies of Trigonidiinae are (TN1) foretibiae with only one apical spur, medioventral in location; (TN2) claws regularly serrated; (TN3) lateral ocelli usually not distinct; (TN4) when present, male mirror most often longer than wide, diamond-shaped, with a rounded false vein; (TN5) shape of ovipositor (including apex). The apomorphies of the Nemobiinae are (NE1) the relative size of hindtibia subapical spurs; (NE2) ectophallic sclerotization not forming an ectophallic arc, but strengthening the base of ectophallic fold;

Fig. 1 .Fig. 2 .

 12 Fig. 1. Morphology of Trigonidiinae: A, B, head of Trigonidium cicindeloides Rambur, 1838

Fig. 3 .

 3 Fig. 3. Male genitalia of Trigonidiinae: A-C, Cyrtoxipha orientalis Bland & Desutter-

Fig. 4 .Fig. 5 .Fig. 6 .Fig. 7 .

 4567 Fig. 4. Trigonidiinae in the field: A, Trigonidium cicindeloides Rambur, 1838, male; B,

Fig. 8 .

 8 Fig. 8. Nemobiinae in the field: A, Nemobius sylvestris (Bosc, 1792), female; B,

  3 in Chintauan-Marquier et al., 2016, clade C. Molecular phylogeny clearly separates two main clades within Trigonidiidae, corresponding to the subfamilies Trigonidiinae Saussure, 1874 and Nemobiinae Serville, 1839 (Chintauan-Marquier et al., 2016). Since the beginning of cricket taxonomy, most authors have separated the taxa close to the genus Trigonidium as a main cricket group (see Brunner von Wattenwyl, 1882 or

Type genus. Trigonidium Rambur, 1838

Diagnosis. Small to very small species (body lengths ranging between 4 and 15 mm, body width less than 3 mm) with strong setae over whole body, and especially on head dorsum and pronotum. Eyes large, protruding or not from head dorsum; fastigium wider to much wider than scape; median ocellus present, lateral ocelli present or absent. Apical and subapical spurs present. Hind basitarsomeres not serrulated, except for one apical inner and one apical outer spine. Males. When present, harp of male stridulatory apparatus crossed by only one vein, longitudinal and oblique; chords closely set and parallel, chords 2 and 3 separated more distally than chord 1. Male genitalia very small, elongated and flat; rami not separated from the pseudepiphallic sclerite; ectophallic apodemes long and very thin; dorsal cavity very small. Females. Fore wing condition variable. Female ovipositor elongated and straight (Nemobiinae), or curved and flattened laterally (Trigonidiinae); shape and length of the apex differing in each subfamily (see infra), except in the smallest species.

Phylogeny and inner classification.

  Brunner von Wattenwyl, 1893 (He and Shen 2020), the black and yellow pattern of the Cuban species Pineronemobus histrionicus(Zayas, 1976 (Yong 2018), or species of Hygronemobius, which exhibit white spots and stripes contrasting with a black body[START_REF] Desutter-Grandcolas | New Nemobiinae crickets from Guianese and Peruvian Amazonia (Orthoptera, Grylloidea, Trigonidiidae)[END_REF][START_REF] Martins | Two new species of Hygronemobius Hebard, 1913 (Orthoptera, Grylloidea, Nemobiinae) from Brazilian Amazon[END_REF]. As for Trigonidiinae, Nemobiinae emit signals at frequencies from 6 to 9 kHz, owing to their small size[START_REF] Paul | Reproductive isolation in three sympatric species of ground crickets (genus Allonemobius)[END_REF][START_REF] Bennet-Clark | Songs and the physics of sound production[END_REF]. However, Nemobiinae calls are usually less powerful than those of Trigonidiinae, more quiet[START_REF] Paul | Reproductive isolation in three sympatric species of ground crickets (genus Allonemobius)[END_REF][START_REF] Ragge | The songs of the grasshoppers and crickets of Western Europe[END_REF]). In the rainforests in Madagascar, for example, they make a continuous background noise in the diurnal soundscape (S. Hugel in press). Nemobiinae usually live in dense, patchy populations and it is often difficult to identify their acoustic emissions as calls proper. High-frequency sounds are produced by Lissotrachelus ferrugineonotatus(He and Shen, 2020) and might be quite common among species with such an incomplete stridulum.As for crickets in general, very few fossil taxa have been described with reasonable certainty as Trigonidiinae or Nemobiinae. Several are based on incomplete and/or poorly preserved fossils (Table1). According to[START_REF] Cigliano | Orthoptera Species File[END_REF], fossil Trigonidiinae include the fossil genus AbanaxiphaVickery & Poinar, 1994 (Abanaxipha incongrua Vickery & Poinar, 1994 and Abanaxipha longispinaVickery & Poinar, 1994, 20.43-13.65 My, Miocene Dominican Gorochov, 2010, 20.43-13.65 My, Miocene Dominican amber); Rhicnogryllus zeuneri[START_REF] Chopard | Orthoptères fossiles et subfossiles de l'ambre et du copal[END_REF] Amusurgus africanus Chopard, 1936 from the Pleistocene East African copal (0.012-0.0 My)

	Acoustic communication. Distribution. Worldwide.
	3.2	Reanalysis of fossil Trigonidiidae

, D), but several genera have evolved toward more specialized habitats, such as caves (as Cophonemobius faustini

Desutter-Grandcolas, 2009

and Hawaiian species of Caconemobius

[START_REF] Kirby | A synonymic catalogue of Orthoptera. Vol. 2. Orthoptera Saltatoria. Part 1. Achetidae and Phasgonuridae[END_REF]

, small cavities in uplifted coral shores (Burcini ssp: Fig.

8C

), mosses growing several meters high in trees (Pteronemobius sp. at Réunion island, S. Hugel, pers. obs.), etc. Many species live alongside rivers and can 'walk' (i.e. 'swim') on the water surface, thanks to their long hind tibial spurs covered with very long setae. Bright colours are uncommon in Nemobiinae, with some notable exceptions such as the contrasting black and orange of Lissotrachelus ferrugineonotatus amber); two species that may belong to the genus Cyrtoxipha Brunner von Wattenwyl, 1873 (Cyrtoxipha (?) electrina

Gorochov, 2010 and Cyrtoxipha (?) illegibilis

  My, Cretaceous) was originally described as a Trigonidiinae, but proved not to be a cricket (see infra). Fossil Nemobiinae include Baltonemobius fossilisGorochov, 2010 (37.2-33.9 My, Baltic amber, Middle Eocene), Pteronemobius (?) anglicus Zeuner, 1937 (Latest Eocene-Earliest Oligocene, Isle of Whight), a young juvenile described as Nemobius sp. by Chopard (1936) from the Middle Eocene Baltic amber, and the mid-Cretaceous Birmaninemobius hirsutus Xu et al., 2020 (but see infra).

  , to Trigonidiinae and Nemobinae. Age according to Fossilworks website (http://fossilworks.org ) and to[START_REF] Shi | Age constraint on Burmese amber based on UePb dating of zircons[END_REF] for Birmaninemobius hirsutus[START_REF] Xu | The first ground cricket (Orthoptera: Trigonidiidae: Nemobiinae) from mid-Cretaceous Burmese amber[END_REF]

	Fossil taxon	Origin	Age (in Ma)	Specimen	Family / Subfamily	Comment
	Abanaxipha	Dominican	Miocene	Nearly complete	Trigonidiidae	Male generic
	longispina	amber	20.43-13.65	female (type)	Subfamily to be	attribution
	Vickery and				checked.	questionable.
	Poinar, 1994
				Male described	Trigonidiidae
				by Gorochov	Trigonidiinae
				(2010)
	A. incongrua	Dominican	Miocene	Incomplete	Trigonidiidae	Generic
	Vickery and	amber	20.43-13.65	female	Trigonidiinae	attribution
	Poinar, 1994					questionable
	Amusurgus	East	Pleistocene	Complete	Trigonidiidae
	africanus	African	0.012-0.0	female	Trigonidiinae
	Chopard, 1936	copal
	Anaxipha	Dominican	Miocene	Three complete	Trigonidiidae	Generic
	dominica	amber	20.43-13.65	females	Trigonidiinae	attribution
	Vickery and					questionable
	Poinar, 1994
	Baltonemobius	Baltic	Middle-Late	Juvenile	Trigonidiidae
	fossilis	amber	Eocene		Nemobiinae
	Gorochov, 2010		37.2-33.9
	Birmaninemobius	Burmese	Mid Cretaceous Complete male	Trigonidiidae	Oldest
	hirsutus Xu et al.,	amber			Trigonidiinae stem	representative
	2020				group	of the family
	? Cyrtoxipha	Dominican	Miocene	Complete	Trigonidiidae	Generic
	electrina	amber	20.43-13.65	female	Trigonidiinae	attribution
	Gorochov, 2010					questionable
	? Cyrtoxipha	Dominican	Miocene	Incomplete	Trigonidiidae	Generic
	illegibilis	amber	20.43-13.65	remains of a	Trigonidiinae	attribution
	Gorochov, 2010			male		questionable
	Liaonemobius	Mandchuria Jurassic	Juvenile	Elcanidae	Not a
	tanae Ren, 1998		125.45-122.46			Gryllidea
	Nemobius sp.	East	Oligocene	Juvenile	Trigonidiidae	Generic
		Prussia	37.2-33.9		Nemobiinae	attribution
						questionable
	Pteronemobius	Isles of	Oligocene	Part of female	?	Uncertain
	(?) anglicus	Wight	37.2-33.9	fore wing		taxonomic
						attribution
	Rhicnogryllus	East	Pleistocene	Complete male	Trigonidiidae
	zeuneri Chopard,	African	0.012-0.0		Trigonidiinae
	1936	copal

Acknowledgments

We thank Stéphanie Boucher (LEM) and George Poinar (University of California, Berkeley) for their help looking for the fossil of Abanaxipha longispina Vickery and Poinar, 1994;Ren Dong and He Tian (College of Life Sciences, Capital Normal University, Beijing, China) for photos of Liaonemobius tanae Ren, 1998. We thank two unknown reviewers for their help improving the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Declaration of competing interests

The authors declare no conflict of interest.


Thomas Girardet

Amel Cherraj

Astrid Pinzano

Christel Henrionnet

Franck Cleymand

Solenne Fleutot
email: solenne.fleutot@univ-lorraine.fr

Study of the influence of autoclave sterilization on the properties of citrate functionalized iron oxide nanoparticles

Keywords: iron oxide nanoparticles, synthesis, functionalization, sterilization, physicochemical properties, magnetic properties

Introduction

For several decades, nanoparticles (NPs) have been used in different fields: depollution [1], energy storage [2], biomedical applications [3][4][5][6][7]. In this latter field, magnetic NPs at room temperature are often used for diagnosis (contrast agent for MRI) [7], for therapy (hyperthermia) [3] or both [4]. In these cases, magnetic NPs must be biocompatible, stable in aqueous solution and have controlled magnetic properties. To achieve these characteristics, magnetic iron oxide nanoparticles are a good candidate [8,9].

There are several types of magnetic iron oxide NPs including SuperParamagnetic Iron Oxide Nanoparticles (SPI-ONs) [7,[10][11][12]. For example, with a superparamagnetic state at room temperature, SPIONs are used as a negative contrast agent for MRI for the lymphatic system [10]. SPIONs are conventionally coated with an organic or an inorganic shell to improve their solubility, stability and biocompatibility [13]. Several studies are based on this stability of the iron oxide nanoparticles in aqueous solution using different organic shells: with polymers such as dextran [14], Poly Ethylene Glycol (PEG) [15], Poly Vinyl Pyrrolidone (PVP) [16], chitosan [17] or with small molecules such as citric acid [18], catechol [19].

For biomedical applications, NPs must be sterilized. Two main methods of sterilization exist: terminal sterilization and aseptic processing. The first one is the most used after NPs synthesis [20,21]. There are several methods of terminal sterilization such as sterile filtration [22], UV irradiation [23], chemical sterilization [24], autoclaving [20].

Sterile filtration is used with nanoparticle sizes smaller than the pore size of filter. With the size of the SPIONs in this work, the nanoparticles would be stuck in the filter because this process leads to an agglomeration. With chemical sterilization, organic products like formaldehyde are often used and induce toxicological problems [24]. Finally, autoclaving and UV irradiation are the most widely used sterilization methods because they are easy and quick to use.

In this study, SPIONs covered by an organic citrate shell to stabilize them in aqueous solution are synthesized by a microwave synthesis method. A step of autoclave sterilization is then performed. A study of the evolution of the phys-icochemical and magnetic properties is carried out before and after treatment to estimate the impacts of these sterilization process.

Experimental

Materials

Ferrous chloride (FeCl2.4H2O) and ferric chloride (FeCl3.6H2O) were purchased from Alfa Aesar. Citric acid (C6H8O7) and ammonium hydroxide solution (NH4OH) were purchased from Sigma Aldrich. In all experiment, ultrapure water (resistivity = 18.2 MΩ.cm) was used.

Synthesis

A mixture of ferric chloride (FeCl3.6H2O; 3.70mmol) and ferrous chloride (FeCl2.4H2O; 5.03 mmol) with citric acid (C6H8O7; 3.16 mmol) are solubilized in 15 mL of ultra-pure water in a Pyrex reactor for the microwave. This solution is stirred until homogenization. Then, 5 mL of ammonium hydroxide solution is added to the mixture to precipitate the solution. The solution turns black and is stirred again before being placed in the microwave to heat at 96 °C for 40 min. A single-mode microwave (Monowave 400 from Anton Paar) is used. The system operates at a frequency of 2.45 GHz. The maximum power of this microwave is 850 W. The temperature is controlled by an external infrared sensor [25].

After heating, the solution is collected and washed by centrifugation. The solution is centrifuged a first time with ethanol at 10,000 rpm for 5 min and a second time with a mixture of ultrapure water and ethanol at 10,000 rpm for 5 min. The nanoparticles (SPIONs) obtained are then dispersed in ultrapure water. A part is evaporated at 60°C to obtain powder for X-Ray Diffraction, ThermoGravimetric Analysis and magnetic measurements. The other part is kept in solution for Transmission Electronic Microscopy, Fourier Transformed InfraRed and Dynamic Light Scattering measurements.

Autoclaving

To sterilize SPIONs, an autoclaving treatment is performed at 120°C during 30 min using an autoclave from Advantage Labo (model AL02-07-100).

Characterization

Different techniques are used for the characterization of nanoparticles.

Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is used to analyse the shape, size and solvent dispersion of nanoparticles. The TEM is a CM200-FEI operating at 200 kW (point resolution 0.27 nm). The size distribution is calculated using free software ImageJ [26].

Dynamic Light Scattering

Dynamic Light Scattering experiments (DLS) were performed by the ZETASIZER Nano ZS device from Malvern Instrumental. The aqueous solution of nanoparticles is diluted one hundred times (final concentration of 0.2 mg/mL) to determine the hydrodynamic size distribution and polydispersity. The isolated or aggregated character of the SPIONs influence the colloidal stability. To evaluate this stability in an aqueous solution, DLS experiments are conducted to determine the average hydrodynamic diameter of the SPIONs in suspension. The DLS measurements are performed three times and the average of these three measurements will be presented in this work.

X-Ray diffraction

X-Ray Diffraction (XRD) patterns were recorded in standard conditions with an INEL CPS120 equipped with a monochromatic cobalt radiation (Co Kα = 0.17886 nm) at grazing angle of incidence simultaneously on 120°. Crystallite size was calculated with the Debye-Scherrer equation from the most intense peak for (311) plane [START_REF] Gravereau | Introduction à la pratique de la diffraction des rayons X par les poudres[END_REF].

Fourier Transform Infra-Red spectroscopy

Fourier Transform InfraRed analysis (FTIR) were performed with a Nicolet 6700 from Thermo-Fisher between 400 and 4 000 cm -1 .

Thermogravimetric analysis

ThermoGravimetric Analysis (TGA) were performed on nanoparticle powder to determine the quantity of the organic shell and of the iron oxide core. The device used was a SETSYS EV 1750 TGA microbalance from Setaram. Experiments were conducted in the temperature range of 20°C to 600°C under air flow (20 mL/min) at a heating rate of 10°C/min.

Magnetic measurements

The NPs magnetic properties in powder state were determined by Superconducting Quantum Interference Device (SQUID) with a Vibrating Sample Magnetometer (VSM) Quantum Design head to determine the saturation magnetization (Ms) and the blocking temperature (TB). The magnetization was measured by sweeping the magnetic field from +5 T to -5T, and then from -5T to +5T.

Magnetization curves as a function of the temperature were recorded as follows: samples were introduced in the SQUID at room temperature (300K) and cooled down to 5 K with no applied field after applying a careful degaussing procedure. A magnetic field of of 200 Oe was then applied, and the magnetization was recorded upon heating from 5 to 300 K (zero field cooled curves called ZFC curves). The sample was then cooled down to 5 K under the same applied field, and the magnetization was recorded upon heating from 5 to 300 K (field cool curve called FC curves).

Results and discussion

As the organic citrate layer used in this study is composed exclusively of carbons, oxygen and hydrogen, only the inorganic core is visualized by TEM. The TEM micrographs in Fig. 1 show spherical and isolated SPIONs before (Fig. 1.a) and after (Fig. 1.b) sterilization by autoclaving.

The narrow size distribution histograms are similar before (Fig. 1.c) and after (Fig. 1.d) sterilization. The average particle diameters determined by ImageJ are 2.10 ± 1.15 nm for 2163 counted nanoparticles and 2.57 ± 0.99 nm for 2008 counted nanoparticles respectively before and after sterilization.

TEM analysis of the sterilized or non-sterilized SPIONs show that the sterilization by autoclave doesn't modify the shape, the size and the aggregation state of SPIONs. Figures 2 present a comparison of size distribution obtained by TEM and DLS. For both, only a population of SPIONs is highlighted. The polydispersity index (PDI) for DLS with small values of 0.29 before treatment and 0.23 after treatment confirms the narrow size distribution obtained by TEM. The mean hydrodynamic diameter corresponds to the maximum of the DLS curve. Figure 2 shows a difference between the diameters determined by TEM on a dry sample and the hydrodynamic diameters measured by DLS in solution. In fact, the hydrodynamic diameter corresponds to the diameter of the mineral shell associated with the thickness of the organic shell and the solvation layer resulting from the interaction between the SPIONs and the aqueous solvent [START_REF] Lim | [END_REF]. The hydrodynamic diameter of the SPIONs before (Fig. 2.a) and after (Fig. 2.b) autoclaving is respectively equal to 8.56 ± 2.76 nm and 8.77 ± 2.80 nm. These measurements confirm the conclusions of the TEM analyses. The autoclaving process doesn't modify the nanoparticles size and doesn't induce an aggregation phenomenon. XRD patterns of SPIONs powder samples show in Fig. 3. Magnetite (Fe3O4) and maghemite (γ-Fe2O3) crystallize in a same cubic crystal structure (space group Fd3m) making it difficult to separate these two structures. Several studies are showed the difference between the XRD pattern of magnetite and of maghemite thanks to the presence of two peaks for the maghemite (corresponding to the appearance of peaks due to vacancies) [6] or thanks to the deconvolution method of some peaks [29].

To determine the influence of sterilization by autoclave on structural properties, the XRD patterns before and after sterilization are compared to a reference of magnetite nanoparticles (JCPDS file: 04-009-2284). The major peaks of the magnetite planes (220), ( 311), (400), ( 511) and ( 440) are present and confirmed the crystallization of the inorganic core. With the Debye Scherrer equation and using the main peak (311), the crystallites size is obtained. The crystallites size is equal to 3.42 nm before sterilization and 3.54 nm after sterilization. The SPIONs sterilization with an autoclave does not influence the crystalline structure. FTIR spectra of SPIONs before and after sterilization, shown in fig 4, can be used to characterize the organic layer and the inorganic core. First, it is important to note that spectra are similar, so the organic layer of SPIONs are not degrade by autoclave sterilization . The large band from 3500 cm -1 to 2500 cm -1 corresponds to the ν(OH) of water. The band at 3200 cm -1 is assigned to the ν(NH) of free ammonium. The band at 2800 cm -1 corresponds to the νs(CH2). The bands at 1610 cm -1 , 1402 cm -1 and 1064 cm -1 are assigned to νa(OCO -), δs(OCO -) and νs(CO) respectively. Finally, the bands at 632 cm -1 and 580 cm -1 corresponds to the elongation of Fe-O bond for maghemite and magnetite respectively [30] . Absorbance Wavenumber (cm -1 )

SPIONs before sterilization SPIONs after sterilization

d s (OCO -) n (OCO -) n (CO) n (NH) n (CH 2 )
Fig. 4: FTIR spectra of SPIONs before (black) and after (red) sterilization between 4000 cm -1 and 400 cm -1 .

To confirm the presence of the organic layer, TGA measurements are realized. The heating curves of SPIONs sample before and after sterilization are shown in Fig. 5. The same behaviour between SPIONs before and after sterilization is observed. In a first time, around 100°C, a weight loss is present and corresponds to the evaporation of water. Then, a second stage from 180°C to 250°C corresponds to the decomposition of citrate. Finally, a last weight loss is produced around 320°C for the last residues of citrate. The remaining mass corresponds to the mass of iron oxide which was oxidized to hematite (α-Fe2O3). For SPIONs before sterilization, the percentage weight loss is equal to 39% (9% of water and 30% of the organic layer): the remaining mass of the powder is equal to 61% and corresponds to the percentage of iron oxide. For SPIONs after sterilization, the powder contains 62% of iron oxide and 31% of organic layer. Both the TGA and FTIR results confirm the non-degradation of organic layer during the sterilization. Indeed, the autoclave treatment temperature of 120°C is lower than the decomposition temperature of the citrate observed by thermogravimetric analysis: the autoclave cannot degrade the citrate layer. The magnetization curves M(H) at 300K and at 5K (with and without a magnetic field during the cooling) are presented in Fig. 6. Without effect of the sterilization, no hysteresis is observed at 300K (Fig. 6.a) which is characteristic of superparamagnetic behaviour. At 300K, Ms are equal 54.61 emu.g -1 and 53.10 emu.g -1 for SPIONs before and after sterilization respectively at 5 T (Fig. 6.a). These values are similar with the MS values of SPIONs which are used as a contrast agent in MRI [31][32][33][34].

At 5K, MS increase and they are equal to 58 emu.g -1 and 61 emu.g -1 for SPIONs before and after sterilization respectively at 0.5 T (Fig. 6.b). These values confirm the same size of SPIONs before and after the sterilization treatment.

To evaluate the potential oxidation at the surface of SPIONs, two magnetization curves are realized at 5K: one without magnetic field during the cooling and the second with a magnetic field (4 T) during the cooling.

For SPIONs before and after sterilization (Fig. 6.c and fig 6.d), the left coercive field is similar with or without applied field during the cooling. Sterilization by autoclaving doesn't influence the superparamagnetic properties of iron oxide nanoparticles. If there is an oxidation at the surface, the magnetic state of the surface will be different compared with the magnetic state of the core and nanoparticles will be core-shell structure defined as Fe3O4@Fe2O3. This core-shell structure would be demonstrated by a difference in values between the left coercive field obtained in the 5K cycle without field and with field. No oxidation at the nanoparticle surface is observed after treatment in accordance with the XRD conclusions. To determine the blocking temperature (TB) defined as the temperature transition between the superparamagnetic state and the ferrimagnetic state, ZFC/FC measurements were recorded (Fig. 7). TB are assimilated to the maximum of ZFC curves and are equal to 14K and 13K before and after sterilization respectively. These values confirm the superparamagnetic state at 300K and the small sizes of the synthesized SPIONs with a size of nanoparticles proportional to the values of TB. In addition, a narrow ZFC curve corresponding to a narrow TB distribution confirms a narrow size distribution of the SPIONs. The narrowness of the ZFC curves before and after autoclave process is in agreement with the narrow size distributions obtained in TEM and DLS.

Conclusions

SPIONs are synthesized by a one-step microwave method. The nanoparticles obtained by this method present a monodisperse distribution with a narrow size distribution, stable in aqueous solution and exhibit superparamagnetic behaviour, which make them good candidates for biomedical applications. Prior to biomedical applications and in vitro contact with cells, an essential step is the sample sterilization. The method used in this study is the autoclave sterilization. A systematic characterization of nanoparticles before and after autoclaving by TEM, DLS, XRD, FTIR, TGA and magnetic measurements has demonstrated that SPIONs keep their physicochemical and magnetic properties after the autoclave treatment. This sterilization treatment doesn't modify the size, morphology or crystal structure of SPIONs. This sterilization does not degrade the organic shell and preserved the surface state of the inorganic core. No additional oxidation is observed. Without aggregation of the nanoparticles after sterilization by this method, the colloidal stability is also preserved. The method of sterilization of SPIONs by autoclave without influence on their physicochemical and magnetic properties is validated before future analysis of cytotoxity and biocompatibility study.

Fig. 1 :

 1 Fig. 1: TEM images of SPIONs (a) before and (b) after sterilization by autoclaving with their size distribution histogram respectively in (c) and (d).

Fig. 2 :

 2 Fig. 2: Comparison between average hydrodynamic diameters determined by DLS measurements (point curve) and with size distribution histogram obtained by TEM of SPIONs (a) before and (b) after sterilization.

Fig. 3 :

 3 Fig. 3: Comparison of XRD patterns of SPIONs before sterilization (black) and after sterilization (red) with XRD pattern of a magnetite NPs reference.

Fig. 5 :

 5 Fig. 5: TGA results of SPIONs before sterilization (black) and after sterilization (red) between 20°C and 600 °C.

Fig. 6 :

 6 Fig. 6: Magnetization curves of SPIONs before (black) and after (red) sterilization as a function of an applied magnetic field at (a) 300K and at (b) 5K. Magnetization curves at 5K without or with applied field during the cooling for SPIONs (c) before sterilization and (d) after sterilization.

Fig. 7 :

 7 Fig. 7: Magnetization as function of the temperature measured under a permanent field of 200 Oe. ZFC-FC curves of SPIONs before (black) and after (red) sterilization.

Thomas Girardet, Amel Cherraj, Astrid Pinzano, Christel Henrionnet, Franck Cleymand, et al.. Study of the influence of autoclave sterilization on the properties of citrate functionalized iron oxide nanoparticles. Pure and Applied Chemistry, 2021, pp.000010151520210303. 10.1515/pac-2021-0303. hal-

Acknowledgments:

We would like to acknowledge the MEM, X-Gamma and Magnetism Competence Centers of Institute Jean Lamour for assistance in TEM, X-ray diffraction and magnetism experiments.