Checklist and taxonomic updates in grasshoppers (Orthoptera: Caelifera) of central and southwestern Tunisia with new records and a key for species identification

ABSTRACT Since the publication of Chopard's 1943 book, Les Orthoptéroides d'Afrique du Nord, the diversity of Orthoptera in Tunisia has not been studied or prospected except for 20 publications. Furthermore, the classification of Orthoptera has changed since 1943 due to taxonomic and phylogenetic advances. To allow a full survey of the Tunisian grasshopper fauna, it is thus necessary first to correctly survey the biological diversity of grasshoppers in Tunisia, and second to have an updated taxonomic reference in order to describe this diversity and compare it with the grasshopper faunas in the other countries of the Maghreb. In the present paper, we propose an updated checklist and a key for the identification of Tunisian grasshoppers, based primarily on field sampling in central and southwestern Tunisia, and literature data for other Tunisian areas. Each species is documented with habitus photographs, geographical distribution, and type of habitat. In total, for the prospected areas, 75 species of Caelifera belonging to five families and 43 genera are listed, while 83 species were recorded up to now for the whole Tunisia. Among these 75 species, seven are newly recorded for Tunisia, i.e., Sphodromerus decoloratus Finot, 1894, Egnatioides coerulans (Krauss, 1893), Dociostaurus biskrensis Moussi & Petit, 2014, Aiolopus puissanti Defaut, 2005, Hilethera aeolopoides (Uvarov, 1922), Leptopternis rothschildi Bolívar, 1913, and Tenuitarsus angustus (Blanchard, 1836); and one species is newly recorded for central and southwestern Tunisia, i.e., Oedipoda fuscocincta fuscocincta Lucas, 1849. We also confirm the presence of two species that were only tentatively recorded in Tunisia, i.e., Oedaleus senegalensis (Krauss, 1877) and Stenohippus mundus (Walker, 1871). DNA sequences (COI, ND2 and H3) are presented for 26 taxa, as a first step towards barcoding all Tunisian caeliferan taxa.


INTrODUCTION
With more than 28000 described species and subspecies distributed worldwide (Cigliano et al. 2020), Orthoptera are considered the most diverse order of polyneopteran insects. This order is divided into two monophyletic suborders: the Ensifera (crickets, katydids and allies) with long antenna (longer than the body and with more than 30 articles), and the Caelifera (grasshoppers and allies) with short antenna (shorter than the body and with less than 28 articles) (Song et al. 2015).
Grasshoppers have been the subject of considerable attention related to their catastrophic damage to crops and all types of green vegetation. North Africa has a long history of desert locust plague upsurges, as it has been and still remains a witness to repeated locust invasions, especially by Schistocerca gregaria (Forskål, 1775).
The earliest scientific expeditions in North Africa started during the colonial period between 1883 and 1884 (Tlili et al. 2019b). Most more recent studies in the Maghreb have been part of field surveys of locust control. The countries that were most vulnerable to locust invasions had the largest share of studies. In that matter, Tunisia is the country with the fewest research studies published on Orthoptera compared to neighbouring countries, e.g. Morocco, Algeria, and Libya. As a result, Tunisia is still poorly known in terms of grasshopper diversity (Louveaux & Ben Halima 1986;Tlili et al. 2019b).
Based on intensive field work, this paper aims at completing the picture of the grasshopper fauna in Tunisia. An updated checklist and taxonomy for grasshoppers is given, limited at this stage to central and southwestern Tunisia, and a key for identification of all genera, species and subspecies is given. In total, 75 species of Caelifera belonging to five families and 43 genera are listed, while only 83 were recorded up to now from the whole country. Among these 75 species, seven are newly recorded for Tunisia, i.e., Sphodromerus decoloratus Finot, 1894, Egnatioides coerulans (Krauss, 1893), Dociostaurus biskrensis Moussi & Petit, 2014, Aiolopus puissanti Defaut, 2005, Hilethera aeolopoides (Uvarov, 1922, Leptopternis rothschildi Bolívar, 1913, andTenuitarsus angustus (Blanchard, 1836); and one subspecies is newly recorded for central and southwestern Tunisia, i.e., Oedipoda fuscocincta fuscocincta Lucas, 1849. We also confirm the presence of two species for which there previ-ously existed only tentative records, i.e., Oedaleus senegalensis (Krauss, 1877) and Stenohippus mundus (Walker, 1871).
Taxonomic studies incorporate an ever-increasing number of characters to identify different taxa, and the use of molecular data is now becoming common practice, even though its usefulness is directly dependent on the number of individuals sampled for the same molecular marker. To facilitate future studies of grasshoppers from the Maghreb we document 26 species for COI, H3 and / or ND2 DNA markers (Table 4).

Sampling area
The present work is part of a study of the grasshopper fauna from central and southwestern Tunisia i.e., Kasserine, Gafsa, Tozeur and Kebili (Fig. 1). Grasshoppers were sampled during four successive years (2016 to 2019) in 16 localities ( Fig. 2; Table 1), with three surveys each year (Tlili et al. 2016). They were actively searched and collected with a sweep net or by hand.  Table 1 for locality coordinates.

Taxonomic liST
Taxa are listed in alphabetical order of the genera and species in each family and subfamily. The nomenclature follows the current classification of the Orthoptera Species File Online (http:// Orthoptera.SpeciesFile.org) (Cigliano et al. 2020). For each species, we give the following information: original taxonomic combination with author, year and page number; successive taxonomic combinations (including synonyms cited in the northern part of Africa, i.e., Lybia, Tunisia, Algeria, Morocco, and Canary Islands) with authors, year and page number; published geographical data for central and southwestern Tunisia; list of examined material; new geographical data resulting from our sampling effort; data on habitat, and remarks. Species distributions were established based on published data (e.g. Dirsh 1965;Usmani 2008;Willemse et al. 2018). In this study, the northern part of Africa, hereafter referred to as North Africa, is limited to Lybia, Tunisia, Algeria, Morocco, and Canary Islands (Tlili et al. in prep.).
In the species list and Table 2, species marked with "*" have been found by previous authors, but not during our field survey; species marked with "**" are new records for Tunisia; species marked with "***" are confirmed in Tunisia. The species marked with a "▼" have been sequenced in the present study.

DaTa from liTeraTure for cenTral anD SouThweSTern TuniSia
We searched all available studies of grasshoppers from 1884 to 2020, in central and southwestern Tunisia ( Fig. 1), but only 20 publications could be found for the Orthoptera. We did not take into account however studies in which the record of species did not refer to specimens deposited in museum collections, which precludes checking identifications (e.g. Doumandji-Mitiche et al. 1990).

maTerial examineD
The collected specimens were first identified using the keys of Chopard (1943) and Dirsh (1965). Identifications were then checked with the interactive key proposed by Louveaux et al. (2020) in the internet database "Orthoptera Acridomorpha from NorthWest Africa" (Louveaux et al. 2020; http:// acrinwafrica.mnhn.fr), and finally confirmed by comparing each specimen with the specimens deposited in the reference collections in the MNHN and NHM.
For the species mentioned in the literature from Tunisia, but not found during our fieldwork, we examined the specimens originating from Tunisia deposited in MNHN and NHM collections. If no specimen from Tunisia was found, we examined samples from neighbouring countries (e.g. Libya, Genitalia images were also taken using a Canon camera EOS 6D attached to a canon macro lens MP-E 65mm f /2,8. collecTion anD knowleDge DaTabaSe webSiTeS Acrinwafrica (Orthoptera Acridomorpha from North West Africa): http://acrinwafrica.mnhn.fr/ DOrSA (Deutsche Orthopteren-Sammlungen): http://www.dorsa.de MNHN collection database: Specimens deposited in the Arthropod collection of the MNHN can be traced with their inventory numbers, MNHN-EO-CAELIFXXXX, in the collection database of the MNHN at the following address, https://science.mnhn.fr/ institution/mnhn/collection/eo/search NHM collection database: Specimens deposited in the NHM can be traced in the Natural History Museum Data Portal at the address, http://data.nhm.ac.uk OSF (Orthoptera Species File): http://Orthoptera.SpeciesFile.org geographic DaTa anD mapS We used QGIS 3.8.0 software to plot the localities sampled on the map of Tunisia. We used the maps of Aubert (1892) to identify the old localities which names have changed over time.

molecular proTocolS
We extracted total genomic DNA from middle or hind femora of dried, alcohol-preserved, or newly collected specimens. We used the QIAamp DNA tissue kit (Qiagen) following the manufacturer's protocol. The molecular work was performed at the Service de Systématique Moléculaire of the MNHN.
We sequence three markers, two mitochondrial and one nuclear, used in previous phylogenetic studies on insects  ( Table 3). These are a fragment of the mitochondrial gene coding for the cytochrome oxidase I (COI, c. 650 bp), a fragment of the mitochondrial gene coding for the NADH dehydrogenase 2 (ND2, c. 400 bp) and a fragment of the nuclear gene coding for the protein H3 (H3, c. 300 bp). Primers and annealing temperatures are given in Table 3. Sequencing reactions were carried out on both DNA strands. Ambiguous results were checked by multiple sequencing either of different DNA extractions from the same individuals or from an extraction from another conspecific individual. The quality of museum-preserved specimens varied considerably and DNA degradation did not allow the amplification of all target sequences for each species (Table 4).
Newly generated sequences were edited in Sequencer v. 4.9 (Gene Codes Corporation, Ann Arbor, MI, USA) and Mesquite 3.6 (Maddison & Maddison 2018) and blasted with NCBI blast tools (Table 4). New sequences have been submitted to GenBank, where they should be published in January 2021.

reSulTS
In this study, we recorded 64% (48 from 75) of species, 76.7% (33 from 43) of genera, 92.8% (13 from 14) of subfamilies, and all the families of grasshoppers mentioned until present for the Tunisian fauna. To these taxa, we added eight new records for this country and confirmed the presence of two additional species (Table 2).
We generated new DNA sequences for 54% (26 from 48) of species, 61% (19 from 33) of genera, 71% (10 from 14) of subfamilies and 80% (4 from 5) of families of grasshoppers recorded in this research for Tunisia (Table 4). The molecular markers amplified are mentioned in the information sheet of each species.      new DaTa for cenTral anD SouThweSTern TuniSia. -We made several surveys specifically for this species specifically, following the paths of the past collectors, but we did not find it.
remarkS. -The last collected specimens of this species are those of Bonnet (1884).
45. Head, pronotum, metanotum and first abdominal tergites with many impressed points; sometimes pronotum covered by a net-work of raised carinulae; aedeagus valves stout, epiphallus with few big spines, and hind border deeply concave (Fig. 97A, B) (Fig. 70). -Head smooth, often with some small white points and a network of carinulae behind the eyes; pronotum covered by more or less wide tubercles, evidently raised; fore and hind borders of the pronotum thick, with white and dark spots; aedeagus valves slender, epiphallus with small spines and hind border concave (Fig. 96A, B) (Fig. 59).
-Prozona with lateral carina; furcal suture of mesosternum slightly curved backward (Fig. 87F) (Fig. 13). -Mesosternal suture straight between mesosternal lobes (Fig. 87B)  58. Inner side of posterior femur with a single large black spot (Fig. 89C)  -Inner side of posterior femur with two small black spots (Fig. 89D)  60. Pronotum posterior margin angular (Fig. 79B) (Fig. 34).    negatively impacted through natural and anthropic changes in the environment, leading potentially to their disappearance, or to their displacement toward other habitat types, or their specialisation to very localized habitats (see Tlili et al. 2019a for Dericorys albidula for example. Further investigation of all types of habitat from Tunisia are now necessary to check this hypothesis. More sampling is thus necessary to increase knowledge of Tunisian grasshoppers. The present paper is meant to facilitate further contributions. We focus here on species morphology, but molecular data should also be generated and compared to help taxonomic identification of taxa for which morphological characters are ineffective or insufficient. This long-term task, which must be based on well-documented and preserved specimens, has been initiated in the present study for species belonging to 19 genera, using three different mitochondrial genes e.g. COI,ND2 and H3 (Tables 3,4). Additional molecular tools, based on high-throughput sequencing of DNA and rNA, are now available to explore the molecular mechanisms underlying morphological biodiversity at varying levels of divergence in African orthopterans (Moussi et al. 2018).