Bruno Le Ru

Claire Capdevielle-Dulac
email: claire.capdevielle-dulac@legs.cnrs-gif.fr

Desmond Conlong
email: des.conlong@sugar.org.za

Beatrice Pallangyo
email: beatricepallangyo@yahoo.com

Johnnie Van Der Berg

George Ong'amo
email: gongamo@uonbi.ac.ke

Gael G Kergoat
email: kergoat@supagro.inra.fr

Johnnie Van Den Berg
email: johnnie.vandenberg@nwu.ac.za

Gael J Kergoat

A revision of the genus Conicofrontia Hampson (Lepidoptera, Noctuidae, Apameini, Sesamiina), with description of a new species: new insights from morphological, ecological and molecular data

Keywords: Conicofrontia, molecular phylogenetics, molecular species delimitation, Sesamiina, systematics, taxonomy

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Introduction

African noctuid stem borers of the tropical subtribe Sesamiina consist of 13 genera encompassing about 200 species [START_REF] Zilli | Apameini. Noctuidae Europaea[END_REF][START_REF] Toussaint | Palaeoenvironmental shifts drove the adaptive radiation of a noctuid stemborer tribe (Lepidoptera, Noctuidae, Apameini) in the Miocene[END_REF], which are usually hardly distinguishable without a thorough examination of wing patterns and genitalia (Moyal & Le Ru 2006;[START_REF] Moyal | Systematics and molecular phylogeny of two African stem borer genera, Sciomesa Tams & Bowden and Carelis Bowden (Lepidoptera: Noctuidae)[END_REF][START_REF] Moyal | Morphological reinforcement, ancient introgressive hybridization and species delimitation in African stem-borer species of the genus Sesamia Guenée (Lepidoptera: Noctuidae)[END_REF][START_REF] Ru | Integrative taxonomy of Acrapex stem borers (Lepidoptera: Noctuidae: Apameini)[END_REF]. About 65% of the species diversity in this subtribe is found in four genera: Acrapex Hampson (more than 80 species), Sesamia Guenée (more than 50 species) [START_REF] Toussaint | Palaeoenvironmental shifts drove the adaptive radiation of a noctuid stemborer tribe (Lepidoptera, Noctuidae, Apameini) in the Miocene[END_REF]. The remaining genera consist of a few species only, as in the case of the genus Conicofrontia [START_REF] Hampson | The moths of South Africa (Part II)[END_REF] described the genus Conicofrontia for Conicofrontia sesamoides based on external characters such as the shape and venation of wings and the structure of palpi, frons and thorax; the genus was named after the slightly conical prominence of the frons. From the beginning, the taxonomic history of Conicofrontia has been a bit confusing as Hampson erroneously associated the male of Speia vuteria (Stoll) (= Phalaena vuteria Stoll) with the female of C. sesamoides in his original description [START_REF] Hampson | The moths of South Africa (Part II)[END_REF]. Later on Hampson provided a new description of the genus and described both sexes of C. sesamoides [START_REF] Hampson | Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum[END_REF]. However as pointed out by [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF], Hampson erroneously altered the original spelling of the name, sesamoides, to sesamiodes. Three species were further described in the genus Conicofrontia by Aurivillius [START_REF] Aurivillius | Wissenschaftliche Ergebnisse der schwedischen zoologischen Expedition nach dem Kilimandjaro, dem Meru[END_REF]) and Hampson [START_REF] Hampson | Descriptions of new genera and species of Noctuidae[END_REF][START_REF] Hampson | Descriptions of New Genera and Species of Amatidae, Lithosidae, and Noctuidae[END_REF]: C. mesophaeae Aurivillius, C. mesoscia Hampson and C. scotochroa Hampson. In 1939, Janse included characters of male genitalia in its redescription of the genus. Major clarifications came with the work of [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF], who completed the description of the genus with characters of male and female genitalia for C. sesamoides. They transferred Arenostola diamesa Hampson to the genus Conicofrontia while transferring C. mesophaeae, C. mesoscia and C. scotochroa to the newly erected genus Sciomesa Tams & Bowden. Tams and Bowden also synonymized C. sesamoides with another species described by Hampson in 1910, Phragmatiphila bipartita Hampson, which was described based on the examination of a single female. [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF] also underlined the close morphological similarity of Conicofrontia with the genera Speia Tams & Bowden and Sciomesa. In 1973, Berio described a new species of Conicofrontia, Conicofrontia dallolmoi from Tanzania, putting up a total of three valid species for the genera. These three valid species (C. sesamoides, C diamesa and C. dallolmoi) were later recorded by [START_REF] Poole | Lepidopterorum Catalogus. New Series. Fasc[END_REF] in his Lepidopterorum catalogus . Until 2004, little was known about Conicofrontia host preferences and ecology as specimens had been only obtained from light trap collections. Starting in 2004, extensive surveys were carried out in several sub-Saharan countries by Bruno Le Ru, collecting noctuid stem borer larvae in infested Poaceae and noctuid stem borer adults with light traps. These surveys enabled us to obtain dozen of adult male and female specimens, belonging to the three recorded Conicofrontia species (C. diamesa, C. sesamoides and C. dallolmoi) and to a new species from Tanzania. We also collected a fifth species from the Republic of South Africa, which is morphologically quite distinctive from other Conicofrontia species. In addition, molecular phylogenetic analyses were conducted on a multi-marker dataset that includes all Conicofrontia species and nine representatives of the subtribe Sesamiina. The resulting phylogenetic framework is further used to implement Poisson-Tree-Processes (PTP) molecular species delimitation procedures [START_REF] Zhang | A general species delimitation method with applications to phylogenetic placements[END_REF], which provide an additional line of evidence to support our conclusions.

Abbreviations: BMNH, Natural History Museum, London, United Kingdom; MCSN, Museo Civico di Storia Naturale, Milan, Italy; MNHN, Muséum National d'Histoire Naturelle, Paris, France; NMK, National Museum of Kenya, Nairobi, Kenya; TMSA, Ditsong Museum: formerly Transvaal Museum of South Africa, Republic of South Africa.

Materials and methods

Sampling and morphological study. Sampling of visually damaged grasses (Poales) in Eastern and Southeastern Africa was conducted over ten years (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014) to collect the larval stages of noctuid stem borers within their wild host-plants ( Le Ru et al. 2006a,b). Larvae were reared on artificial diet [START_REF] Onyango | Continuous rearing of the maize stem borer Busseola fusca on an artificial diet[END_REF] until pupation and emergence of adults (Le Ru et al. 2006a,b). Some adult specimens were also collected in light traps. 536 larvae belonging to the Conicofrontia genus were sampled in the localities listed in Table 1. In addition, 40 adults were collected from a light trap set up in Tanzania. The study is based on 87 adult specimens belonging to five species collected in 15 localities in two countries, South Africa and Tanzania. Plant specimens were identified by Kathleen Gordon-Gray (Pietermaritzburg University, South Africa) and Simon Mathenge (Botany Department, University of Nairobi, Kenya).

Genitalia were dissected after immersion of the end of the abdomen in a boiling 10% potash bath for a few minutes, then cleaned, immersed in absolute alcohol for a few minutes and mounted on slides in Euparal (after separating the aedeagus from the rest of the (male) genitalia. Collected insects were identified by comparison with types and specimens housed by BMNH the MCSN and the TMSA. The holotype of the new species was deposited in the MNHN, whereas paratypes were deposited in the MNHN and in the NMK.

DNA extraction and sequencing. For this study, 21 Conicofrontia specimens (sensu Poole 1989) were selected for the molecular analyses, including two specimens from the new species collected in Tanzania. In addition, we included representatives of five other genera in the subtribe Sesamiina as outgroups based on the results of a recent molecular study [START_REF] Toussaint | Palaeoenvironmental shifts drove the adaptive radiation of a noctuid stemborer tribe (Lepidoptera, Noctuidae, Apameini) in the Miocene[END_REF]. We also used a specimen of the more distantly related genus Spodoptera (Mitchell et al. 2006;[START_REF] Toussaint | Palaeoenvironmental shifts drove the adaptive radiation of a noctuid stemborer tribe (Lepidoptera, Noctuidae, Apameini) in the Miocene[END_REF] to root the tree. DNA was extracted from hind legs using Qiagen DNAeasy tissue kits (Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) amplifications were conducted for four mitochondrial gene fragments, a 658 bp region of the cytochrome oxidase I (COI), 976 bp of the cytochrome b (Cytb), 360 bp of the ribosomal 12S RNA (12S), and 566 bp of the ribosomal 16S RNA (16S). Two nuclear gene regions were also sequenced, 848 bp of the 28S ribosomal DNA (28S), and 1,240 bp of the elongation factor-1a (EF1a). For both genes, we used the primers and settings detailed in [START_REF] Kergoat | Disentangling dispersal and vicariance patterns in armyworms: evolution and historical biogeography of the pest genus Spodoptera (Lepidoptera: Noctuidae)[END_REF]. Resulting PCR products were processed by the French sequencing center Genoscope using a BigDye v3.1 sequencing kit and Applied 3730xl sequencers. Both strands were sequenced for all specimens to minimize PCR artefacts and ambiguities. Sequences of complementary strands were edited and reconciled using Geneious v5.1 software (available at: www.geneious.com/). All the sequences generated in this study were deposited in GenBank (see Appendix S1 for the accession numbers). Unlike the sequences of coding genes (COI, Cytb, and EF1a), the sequences of ribosomal genes (12S, 16S and 28S) were variable in length. Their alignment was accomplished using MAFFT 7 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF] with default option settings. For all protein-coding genes, we used Mesquite 3.0 (available at: www.mesquiteproject.org) to check the coding frame for possible errors or stop codons. The combination of the six gene fragments resulted in a combined matrix of 31 specimens and 4,648 aligned characters.

Phylogenetic and molecular species delimitation analyses. Phylogenetic analyses were conducted using Maximum likelihood (ML), as implemented in RAxML v8 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF]. We used partitioned analyses to improve phylogenetic accuracy [START_REF] Nylander | Bayesian phylogenetic analysis of combined data[END_REF]. Partitions and substitution models were determined using PartitionFinder v1.1.1 (Lanfear et al. 2012). The corrected Akaike information criterion (AICc; Posada & Buckley 2004) was used as a metric for ML analyses. Based on the AICc results we used 12 partitions with either a General time reversible (GTR)+G+I model or a GTR+G model (see Appendix S2). The best tree was obtained using a heuristic search implementing 100 random-addition replicates. Clade support was then assessed using nonparametric bootstrap values (BV) (1,000 replicates were used). Nodes supported by BV > 70% were considered as strongly supported following [START_REF] Hillis | An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis[END_REF].

Poisson-tree-processes molecular species delimitations were conducted on the Web server of the Exelixis Lab (http://species.h-its.org/ptp/), with default settings. With the PTP model, speciation or branching events are modelled in terms of number of substitutions (represented by branch lengths), so it only requires a phylogenetic input tree. The corresponding analyses were carried out using the best tree from the ML analyses.

Results

Taxonomy

After having cross-checked against types preserved in the Museum to avoid coincidence of synonymies we provide morphological evidence to support the transfer of C. dallolmoi to Hygrostola Warren. We also establish that P. bipartita sensu Hampson is in fact a valid species, distinct from C. sesamoides; specimens of this species are in fact represented in our sampling and correspond to the fifth species, from the Republic of South Africa. We present the description of the new species C. lilomwi nov. sp. from Tanzania. We also provide a supplemental description of the Conicofrontia genus and of the previously described species, with male and female genitalia presented for the first time for all species.

Genus description. Conicofrontia Hampson, 1902:296. Type species: Conicofrontia sesamoides [START_REF] Hampson | The moths of South Africa (Part II)[END_REF]Conicofrontia Hampson, 1910:338 (redescription); Conicofrontia Hampson, Janse, 1939: 379-383 (redescription, with characters of male genitalia); Conicofrontia Hampson, Tams & Bowden, 1953:651 (redescription, with characters of male and female genitalia).

The description by [START_REF] Hampson | The moths of South Africa (Part II)[END_REF] and further redescriptions [START_REF] Hampson | Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum[END_REF][START_REF] Janse | The Moths of South Africa[END_REF][START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF] of the genus Conicofrontia are confirmed by the re-examination of morphological characters. The genus has homogeneous habitus and genitalia. In all known species, palpi upturned, porrect in both sexes; frons with a slightly conical prominence in both sexes; antenna of male are serrate while those of female are simple; forewings with a longitudinal median brown streak most of the time, particularly marked in males. The forewings present colour, pattern and shape variation in all four species with males being darker than females and with more contrasted forewing pattern; the rather narrow forewing characteristics underlined by [START_REF] Hampson | The moths of South Africa (Part II)[END_REF][START_REF] Hampson | Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum[END_REF], [START_REF] Janse | The Moths of South Africa[END_REF] and [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF] is supported by our morphological study, for female only; the male forewing is less elongated than the female one, the apex rounded with the termen evenly curved not crenulated; all other characters are confirmed. [START_REF] Hampson | Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum[END_REF]. The abbreviation ZAF is used for Republic of South Africa.

The most characteristic features of male genitalia are the sacculus with a heavily sclerotized and dentate clavus and the presence of a heavily sclerotized and dentate plate across the upper edge. Other characters are tegumen with medium-size peniculi, uncus long, pointed apically; in contrast with [START_REF] Janse | The Moths of South Africa[END_REF] and [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF], we would describe vinculum with well-developed saccus, instead of small; valve elongate and narrow with a narrow cucullus rather rounded at the apex and costal margin with a small sclerotized ridge-like expansion; juxta plate like or oblong pear-shaped with a neck. No clasper, aedeagus short and stout, manica with two-lobed [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF] for C. sesamoides is insufficient. Additional description: bursa copulatrix large and membranous, with one signa; ductus bursae broad and strongly sclerotized throughout and sharply differentiated from the bursa; ductus seminalis from the base of the bursa; ostium bursae with strongly sclerotized ventral lip, the ostial segment membranous throughout. Ovipositor lobes short and broad with many stout bristles in addition to the small setae and an apical crest of short stout bristles.

Conicofrontia bipartita [START_REF] Hampson | Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum[END_REF] Le Ru,comb. nov.,3a,3e,3i,4a) Phragmatiphila bipartita [START_REF] Hampson | Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum[END_REF] Redescription. The female holotype was poorly described by [START_REF] Hampson | Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum[END_REF]. The male is described here for the first time; externally it looks very similar to the female, however the general shape of the female's forewing is more elongated at the apex than that of the male. Additions to the previous descriptions (Figs. 1a-1d): antennae ochreous, filiform in female, serrate in male, cilia short, fasciculate, flagellum adorned dorsally with brown scales, palpus brown. Head and front of thorax ochreous brown, tegulae bright ochreous, legs brown ochreous, abdomen greyish ochreous. Forewing: a longitudinal dark brown fascia from the base along the lower margin of the cell partly within the cell, partly without, extending to just before the termen; the cell ochreous, all other areas (costa, apex, termen and inner margin) ochreous suffused with brown scales; A small subterminal black spot on vein 5, all veins towards the apex adorned with ochreous scales. Fringe ochreous adorned successively with a narrow basal black line, a narrow ochreous line and a thick light brown line. Hindwing; grey strongly tinged with brown veins suffused with brown scales; fringe grey with a narrow basal grey-white line highlighted at the base with a narrow brown line. Underside of the forewing uniformly ochreous; slightly suffused with brown scales on the costa and apex, strongly suffused with brown scales from the cell to the termen, less suffusion in the inner margin. Underside of hindwing ochreous uniformly suffused with brown scales but more densely on costa and apex; veins of both forewing and hindwing adorned with ochreous scales.

Wingspan 35 mm (1 male), 34 mm (1 female).

Male genitalia (Figs. 3a,3e). Uncus long and wide, tapering to a fine point and tufted with long hair on the upperside; tegumen with medium-size rounded peniculi, vinculum with medium-size triangular saccus; valves elongate and narrow; cucullus elongate, rounded at apex and tufted with medium size hairs; sacculus with an heavily sclerotized and dentate clavus, all dents with the same size; the presence of a sclerotized and slightly dentate plate, pear shaped, across the upper edge of the sacculus, costal margin with a small sclerotized ridge-like expansion roundly pointed and slightly curved inwardly; the juxta oblong, elongated pear-shaped without sclerotization with a long and narrow neck shortly bifid. Aedeagus short, slightly curved, manica with a two-lobed sclerotization, less than one fifth length of the aedeagus, ending in a Spinoza tip; vesica with two ventral cornuti slightly dented, one-quarter length of the aedeagus.

Female genitalia (Fig. 3i). Unfortunately the bursa copulatrix can not be described as it has been spoiled during the genitalia preparation; ductus bursae broad and strongly sclerotized on the ostium side; ventral plate of ostium bursae sclerotized bilobate with small lobes funnel-shaped; dorsal plate large, broad, weakly sclerotized. Ovipositor lobes relatively long and narrow (2.5 times longer than wide) with many stout bristles in addition to the small setae and an apical crest of short stout bristles.

Larvae L5 instar (Fig. 4a): length, 35-40mm, width, 4.0 mm; head smooth, orange brown, prothoracic shield pale yellow; body with ground colour buff, dorsally suffused with pink, pinacula pale yellow and caudal plate brown. Young larvae are very similar in appearance to mature ones. Conicofrontia bipartita: 1a-male upper side, 1b-male under side, 1c-female upper side, 1d-female under side. Conicofrontia diamesa: 1e-male upper side, 1f-male under side, 1g-female upper side, 1h-female under side. Conicofrontia lilomwa: 2a-male upper side, 2b-male under side, 2c-female upper side, 2d-female under side. Conicofrontia sesamoides: 2e-male upper side, 2f-male under side, 2g-female upper side, 2h-female under side. FIGURE 3. Genitalia of Conicofrontia species. Scale bar = scale bar = 1 mm and 0.5 mm for male penis. Conicofrontia bipartita: 3a-male genitalia, 3e-male penis, 3i-female genitalia. Conicofrontia diamesa: 3b-male genitalia, 3f-male penis, 3j-female genitalia. Conicofrontia lilomwa: 3c-male genitalia, 3g-male penis, 3k-female genitalia. Conicofrontia sesamoides: 3d-male genitalia, 3h-male penis, 3l-female genitalia. Bionomics. Conicofrontia bipartita is a markedly hygrophilous species inhabiting grasses along banks of streams, rivers and marshes. Second and third instar larvae were collected at the bottom of Miscanthus capensis inflorescence stems, always gregarious. Typically, plants exhibiting signs of infestation by C. bipartita larvae have a dry, brown inflorescence. We suspect that the larvae disperse when they reach the fourth instar. No pupae were found in stems, and therefore borers probably pupate in the soil.

Distribution. South Africa. The two records are from Afromontane (Mosaic no 19) vegetation mosaic [START_REF] White | The vegetation of Africa, a descriptive memoir to accompany the UNESCO / AETFAT / UNSO vegetation map of Africa[END_REF]) (Fig. 6).

Remarks. Easily separated from C. sesamoides with which it has been confused by [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF]. The juxta is plate-like in C. sesamoides while it is pear-shaped in C. bipartita, with the valves broader at basal half in C. bipartita compared to C. sesamoides and the clavus much more elongated toward the apex in C. sesamoides.

Conicofrontia diamesa (Hampson, 1920) (Figs. 1e-1h, 3b, 3f, 3j, 4b) Arenostola diamesa [START_REF] Hampson | On new genera and species of Lepidoptera Phalaenae, with the characters of two new families[END_REF] Redescription. The male holotype was poorly described by [START_REF] Hampson | On new genera and species of Lepidoptera Phalaenae, with the characters of two new families[END_REF]. The male looks very similar to the female, however the general shape of the female's forewing is more elongated at the apex. Additions to the previous descriptions (Figs. 1e-1h): antennae ochreous, filiform in female, serrate in male, cilia short, fasciculate, flagellum adorned dorsally with brown scales, palpus brown, eyes red brown. Head and front of thorax ochreous brown, tegulae bisque, legs brown ringed with bisque, abdomen brown ochreous, anal tuft ochreous. Forewing: a longitudinal brown fascia suffused with fuscous scales from the base along the lower margin of the cell partly within the cell, partly without, extending to just before the termen; the cell, costa, apex, termen and inner margin bisque with areas between veins suffused with fuscous scales in apex and termen; 2-3 small black spots along the upper margin of the cell, sometimes one additional black spot in front of the cell. Fringe bright ochreous or bisque adorned successively from the base with a narrow basal brown line, a thick bisque line and a thick brown line. Hindwing; grey bistre strongly suffused with brown scales; fringe bistre with a narrow grey bistre line at the base. Underside of the forewing bistre suffused with brown and fuscous scales on the costa and apex, strongly suffused with brown scales from the cell to the termen, less suffusion in the inner margin. Underside of hindwing grey uniformly suffused with brown and fuscous scales but more densely on costa and apex; veins of both forewing and hindwing adorned with fuscous scales.

Wingspan 24-27 mm (males) (n = 6); 26-28 mm (females) (n = 6).

Male (27,26,25,25,24,26),Female (27,28,26,26,27,27) Male genitalia (Figs. 3b,3f). Uncus long and wide, tapering to a fine point and tufted with long hair on the upperside; tegumen with medium-size rounded peniculi, vinculum with medium-size triangular saccus; valves elongate and narrow, cucullus elongate, rounded at apex and tufted with medium size hairs; sacculus with an heavily sclerotized and dentate clavus, dents larger at both ends; the presence of a sclerotized and heavily dentate plate across the upper edge of the sacculus, costal margin with a small sclerotized ridge-like expansion roundly pointed and slightly curved inwardly; the juxta plate like, short and becoming smaller towards the top. Aedeagus short, slightly curved, manica with a two-lobed sclerotization, less than one fifth length of the aedeagus, ending in a spinose tip; vesica with two strongly dentate ventral cornuti, almost one third length of the aedeagus.

Female genitalia (Fig. 3j). Corpus bursae long and cylindrical with one signa; ductus seminalis from the basal part of the bursa; ductus bursae broad and strongly sclerotized on the ostium side; ventral plate of ostium bursae sclerotized bilobate deeply invaginated at middle, thus forming a circular-shaped ventral wall of antrum; dorsal plate large, broad, weakly sclerotized. Ovipositor lobes short and wide (1.5 times longer than wide) with many stout bristles in addition to the small setae and an apical crest of short stout bristles.

Larvae L5 instar (Fig. 4b): length, 30-35mm, width, 3.5 mm; head smooth, orange brown, prothoracic shield yellow orange; body with ground colour buff, dorsally suffused with pink, pinacula pale yellow and caudal plate brown. Young larvae are very similar in appearance to mature ones.

Bionomics. Conicofrontia diamesa is a hygrophilous species inhabiting grasses along banks of streams, rivers and marshes. Larvae were collected at the bottom of young stems of Cymbopogon sp. stems, always solitary. Typically, plants exhibiting signs of infestation by C. diamesa larvae have a curled, brown, central leaf. Damaged stems had a small hole (ca. 2 mm diameter) located approximately 10 cm from ground level. We suspect that the larvae disperse when they reach the fourth instar. No pupae were found in stems, and therefore borers probably pupate in the soil.

Distribution. South Africa. The two records are from Afromontane (Mosaic no 19) vegetation mosaic [START_REF] White | The vegetation of Africa, a descriptive memoir to accompany the UNESCO / AETFAT / UNSO vegetation map of Africa[END_REF]) (Fig. 6).

Remarks. Easily separated from other Conicofrontia species with the short juxta plate-like, the dentate clavus with dents larger at both ends, the two strong dentate ventral cornuti of the vesica and the ventral plate of ostium bursae, sclerotized bilobate, deeply invaginated at middle, thus forming a circular-shaped ventral wall of antrum. Description. The male looks brighter the female, and the general shape of the female's forewing is more elongated at the apex than that of the male (Figs. 2a-2d). Antennae ochreous, filiform in female, serrate in male, cilia short, fasciculate, flagellum adorned dorsally with brown scales, palpus brown. Male: head and front of thorax ochreous, tegulae bright ochreous legs brown, abdomen grey ochreous. Forewing: a longitudinal brown fascia from the base along the lower margin of the cell partly within the cell, partly without, extending to just before the termen; the cell bistre, all other areas (costa, apex, termen and inner margin) ochreous suffused with fuscous and bistre scales; a small subterminal black spot on vein 5, a postmedial row of 3-4 black spots between the veins, all veins towards the apex adorned with bistre scales. Fringe bistre adorned with a narrow basal brown line. Hindwing; grey strongly suffused with brown scales; fringe bistre with a narrow basal brown line. Underside of the forewing uniformly brown chocolate, all veins adorned with ochreous scales. Underside of hindwing, grey uniformly suffused with brown scales but more densely on costa and apex; veins adorned with ochreous scales. Female: head and front of thorax brown chocolate, tegulae ochreous, legs brown, abdomen grey ochreous. Forewing: a longitudinal brown fascia from the base along the lower margin of the cell partly within the cell, partly without, extending to just before the termen; the cell dark ochreous, all other areas (costa, apex, termen and inner margin) dark ochreous suffused with brown scales; a small subterminal black spot on vein 5, a postmedial row of 3-4 black spots between the veins, all veins towards the apex adorned with ochreous scales. Fringe ochreous adorned with a narrow basal brown line. Hindwing; grey strongly suffused with brown scales; fringe grey ochreous with a narrow basal brown line. Underside of the forewing grey ochreous strongly suffused with brown scales, all veins adorned with ochreous scales. Underside of hindwing, grey uniformly suffused with brown scales but more densely on costa and apex; veins adorned with brown scales.

Conicofrontia lilomwa

Wingspan 24-26 mm (males) (n = 10); 25-29 mm (females) (n = 9). Male Male genitalia (Figs. 3c,3g). Uncus long and wide, tapering to a fine point and tufted with long hair on the upperside; tegumen with medium-size rounded peniculi, vinculum with medium-size triangular saccus; valves elongate and narrow, cucullus elongate, rounded at apex and tufted with medium size hairs; sacculus with a small sclerotized clavus without dent; the presence of a sclerotized and pear shaped plate across the upper edge, costal margin with a small sclerotized ridge-like expansion roundly pointed and slightly curved inwardly; the juxta short and pear-shaped. Aedeagus short, slightly curved, manica with a two-lobed sclerotization, less than one fifth length of the aedeagus, ending in a spinose tip; vesica with two dentate ventral cornuti, almost one fifth length of the aedeagus.

Female genitalia (Fig. 3k). Corpus bursae long and cylindrical with one signa; ductus seminalis from the basal part of the bursa; ductus bursae broad and strongly sclerotized on the ostium side; ventral plate of ostium bursae sclerotized bilobate with medium size lobes bean shaped; dorsal plate large, broad, weakly sclerotized. Ovipositor lobes short and wide (2 times longer than wide) with many stout bristles in addition to the small setae and an apical crest of short stout bristles.

Larvae L5 instar (Fig. 4c): length, 30-35 mm, width, 3.5 mm; head smooth, black, prothoracic shield dark brown; body with ground colour buff, dorsally suffused with pink, pinacula and caudal plate black. Young larvae are very similar in appearance to mature ones.

Etymology. Named after the village of Lilomwa in Tanzania.

Bionomics. Conicofrontia lilomwa is a markedly hygrophilous species inhabiting grasses along banks of streams, rivers and marshes. Larvae were collected at the bottom of young stems of Cymbopogon giganteus Chiov. Typically, plants exhibiting signs of infestation by C. lilomwa larvae have a curled, brown, central leaf. We suspect that the larvae disperse when they reach the fourth instar. No pupae were found in stems, and therefore borers probably pupate in the soil.

Distribution. Tanzania. The four recorded localities are from Afromontane (Mosaic no 19) vegetation mosaic [START_REF] White | The vegetation of Africa, a descriptive memoir to accompany the UNESCO / AETFAT / UNSO vegetation map of Africa[END_REF]) (Fig. 6).

Remarks. Easily separated from other Conicofrontia species with the short juxta pear shaped, the small clavus without dent, and the ventral plate of ostium bursae, sclerotized bilobate bean-shaped.

Conicofrontia sesamoides Hampson, 1902 (Figs. 2e-2h, 3d, 3h, 3l, 4d) Conicofrontia sesamoides Hampson, 1902:296. Conicofrontia sesamiodes (ex errore) Hampson, 1910:338 (recte sesamoides in Tams & Bowden, 1953:651).

Type material. Holotype (female) of Conicofrontia sesamoides: South Africa: Eastern Cape formerly Cape Colony, Transkei, no locality name, no collection date, Agrotidae genitalia slide 1212, 1947-345 [BMNH]-345 [BMNH].

Other material: South Africa: Redescription. The female holotype [START_REF] Hampson | The moths of South Africa (Part II)[END_REF]) and the male have been poorly described. Like for other Conicofrontia spp., the general shape of the female's forewing is more elongated at the apex than that of the male. Additions to the previous descriptions (Figs. 2e-2h): antennae ochreous, filiform in female, serrate in male, cilia short, fasciculate, flagellum adorned dorsally with brown scales, palpus ochreous. Head and front of thorax ochreous brown, tegulae bisque, legs brown ringed with bisque, abdomen bisque suffused with brown scales. Forewing: male: a longitudinal dark brown fascia from the base along the lower margin of the cell partly within the cell, partly without, extending to just before the termen; the cell bisque, all other areas (costa, apex, termen and inner margin) bisque suffused with brown scales; a small subterminal black spot on vein 5, a postmedial row of 3-4 black spots between the veins, all veins towards the apex adorned with bisque scales. Fringe bisque adorned successively with a narrow basal grey line, a thick bisque line and a thick brown line. Hindwing; grey strongly suffused with brown scales; fringe bisque with a thick wide basal line highlighted in the middle with a narrow brown line. Underside of the forewing buff, costa, apex and inner margin bisque suffused with brown scales, less suffusion in the inner margin. Underside of hindwing bisque suffused with brown scales but more densely on costa and apex. The wing pattern of the female is very similar but all colours brighter.

Wingspan 24-27 mm (males) (n = 9); 26-30 mm (females) (n = 9). Male Male genitalia (Figs. 3d,3h). Uncus long and wide, tapering to a fine point and tufted with long hair on the upperside; tegumen with medium-size rounded peniculi, vinculum with medium-size triangular saccus; valves elongate and narrow; cucullus elongate, rounded at apex and tufted with medium size hairs; sacculus with an heavily sclerotized and dentate clavus pointed to the apex, all dents with the same size; the presence of a sclerotized and heavily dentate plate across the upper edge of the sacculus, costal margin with a small sclerotized ridge-like expansion roundly pointed and slightly curved inwardly; the juxta plate-like narrowing at the tip. Aedeagus short, slightly curved, manica with a two-lobed sclerotization, less than one fifth length of the aedeagus, ending in a spinose tip; vesica with two small dentate ventral cornuti, less than one sixth length of the aedeagus.

Female genitalia (Fig. 3l). Corpus bursae long and cylindrical with one signa; ductus seminalis from the basal part of the bursa; ductus bursae short, broad and strongly sclerotized on the ostium side; ventral plate of ostium bursae sclerotized bilobate deeply invaginated at middle, thus forming an oval-shaped ventral wall of antrum; dorsal plate large, broad, weakly sclerotized. Ovipositor lobes short and wide (2.5 times longer than wide) with many stout bristles in addition to the small setae and an apical crest of short stout bristles.

Larvae L5 instar (Fig. 4d): length, 30-35mm, width, 3.5 mm; head smooth, red brown, prothoracic shield orange brown; body with ground colour buff, dorsally suffused with pink, pinacula and caudal plate orange brown. Young larvae are very similar in appearance to mature ones.

Bionomics. Conicofrontia sesamoides is a markedly hygrophilous species inhabiting grasses along banks of streams, rivers and marshes. Larvae were collected at the bottom of young stems Miscanthus capensis stems, always solitary. Typically, plants exhibiting signs of infestation by C. sesamoides larvae have a curled, brown, central leaf. Damaged stems had a small hole (ca. 2 mm diameter) located approximately 10 cm from ground level. We suspect that the larvae disperse when they reach the fourth instar. No pupae were found in stems, and therefore borers probably pupate in the soil. Infestation of sugar cane fields, Saccharum officinarum Linnaeus, was also recently observed in the Republic of South Africa by Bruno Le Ru (Y. Assefa. pers. com.).

Distribution. Republic of South Africa. The ten records are from Afromontane (Mosaic no 19) and Afromontane related (Mosaic n° 20, 24, 48) vegetation mosaics [START_REF] White | The vegetation of Africa, a descriptive memoir to accompany the UNESCO / AETFAT / UNSO vegetation map of Africa[END_REF]) (Fig. 6).

Remarks. Easily separated from other Conicofrontia species with the long juxta plate-like, dentate clavus pointed to the apex and the ventral plate of ostium bursae sclerotized bilobate deeply invaginated at middle, thus forming an oval-shaped ventral wall of antrum.

Hygrostola dallolmoi (Berio) Le Ru, Conicofrontia dallolmoi Berio, 1973:143. Type material. Holotype (male) of Conicofrontia dallolmoi: Tanzania: Kipengere Mountain, Ikonda, 19-xii-1970, Prep. Berio N°4974 [MCSN].

Other material: Tanzania: 1♂, 1♀ Redescription. The male holotype was poorly described by [START_REF] Berio | Nuove species e generi di noctuidae africane e asiatiche e note sinonimiche. Parte II[END_REF]. The female is described here for the first time; it looks very similar to that of the male, however a little bit larger (Figs. 5a-5d): antennae ochreous, filiform in female, serrate in male, cilia short, fasciculate, flagellum adorned dorsally with ochreous scales, palpus brown, eyes brown. Head and front of thorax ochreous brown, tegulae ochreous, legs fuscous with brown scales, ground colour of abdomen ochreous adorned with a dense suffusion of brown scales on the top particularly in the male. Forewing: a longitudinal thin and short brown fascia from the base along the lower margin of the cell, defined by the greatly indented sub-basal and antemedian veins. The cell ochreous, all other areas (costa, apex, termen and inner margin) ochreous suffused with brown scales; a black spot at the base of the fascia just on the subbasal vein, antemedian vein adorned with brown scales. Fringe bistre adorned successively with a narrow basal ochreous line, and brown spots, variable in number, between the veins. Hindwing: ground colour ochreous strongly suffused with brown scales; an oblique longitudinal fuscous fascia between the posterior cubital and the 1A+2A anal veins, the anal area adorned with dark ochreous, a brown diffuse spot at the apex of the cell; fringe bistre adorned with a broken and brown broad basal line. Underside of forewing bright ochreous slightly suffused with brown scales in the costa, termen and inner margin areas, strongly suffused with brown scales in the apex and cell areas, two brown spots in the cell, a post-medial series of brown elongated marks on the veins, fringe bistre adorned with brown spots, variable in number, between the veins; Underside of hindwing uniformly suffused with brown scales, one brown spot in the cell, a post-medial series of brown elongated marks on the veins, fringe bistre adorned with brown spots, variable in number, between the veins.

Wingspan 53-56 mm (males) (n = 5); 57-58 mm (females) (n = 3). Male (53,56,55,54,54),Female (57,58,58) Male genitalia (Figs. 5e,5f). Uncus long and thin, rounded at the apex and tufted with medium size hair on the upperside; tegumen with medium-size rounded peniculi, vinculum with medium-size rounded saccus, with a pronounced u-shaped indentation at the top margin; valves elongate and narrow; strong cucullus, club-shaped, with a short neck tufted with bristles; sacculus without clavus, presence of a sclerotized plate across the upper edge of the sacculus, costal margin with a small sclerotized ridge-like expansion roundly pointed and slightly curved inwardly; juxta small and flattened. Aedeagus short, curved, without cornuti, presence of a sclerotized elongate like carina crest, vesica armed with many rows of short stout spines. FIGURE 5. Adults and genitalia of Hygrostola dallolmoi. Scale bar = 10 mm for adults, 2 mm for male genitalia, 1 mm for male penis and 3 mm for female genitalia. Hygrostola dallolmoi: 5a-male upper side, 5b-male under side, 5c-female upper side, 5d-female under side, 5e male genitalia, 5f-male penis, 5g-female genitalia. Remarks. Easily separated from Conicofrontia spp. with an uncus rounded apically, a broad club-shaped cucullus, a very small and flattened juxta, absence of two-lobed sclerotization on the manica, no cornuti, elongated ovipositor, no signa.

Phylogenetic and molecular species delimitation analyses. The ML analyses yield a best ML tree with a likelihood score of -14326.10 (Fig. 7). Overall the tree is well supported and all nodes leading to putative taxa (i.e. morphospecies) have BV > 70%. Regarding the five species of interest, our analyses underline that Conicofrontia sensu [START_REF] Poole | Lepidopterorum Catalogus. New Series. Fasc[END_REF] is paraphyletic because of the position of H. dallolmoi, which appears phylogenetically unrelated to the other Conicofrontia species. Specimens belonging to the Conicofrontia genus constitute a well-supported clade (BV of 100%). Within this clade it is interesting to note that specimens corresponding to C. bipartita and C. sesamoides are not closely related. Conicofrontia sesamoides is sister to a clade grouping C. bipartita and C. diamesa; these three species which are distributed in the Republic of South Africa are sister to the last species, C. lilomwa from Tanzania. Regarding the PTP molecular species delimitation analyses, the putative molecular species clusters are completely consistent with the morphological identification.

Discussion

Systematics. The four species treated here make up a morphologically and ecologically homogeneous group, confirming the Conicofrontia genus redescription made by [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF]. The important amount of specimens available for this study, with both sexes available for the five species, allows to precise and complete the Conicofrontia genus description and clarify the confusing taxonomic status of C. sesamoides and Phragmatiphila bipartita, the last one considered as synonym of C. sesamoides by [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF]. The integrative taxonomy developed in this study provides clear morphological, ecological and genetics evidences, allowing us to confirm the status of C. bipartita bona species. The confusion made by [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF] in their revision is most probably the consequence of the scarce material available at that moment. As anticipated by [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF], our study confirms that Arenostola diamesa belongs to the Conicofrontia genus; however if the wing pattern is very close to that of C. sesamoides [START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF], the phylogeny show that C. diamesa is more related to the C. bipartita. On the other hand, both morphology and phylogeny results provide evidences that C. dallolmoi described by [START_REF] Berio | Nuove species e generi di noctuidae africane e asiatiche e note sinonimiche. Parte II[END_REF] does not belong to the Conicofrontia genus, but to the Hygrostola Warren genus. Like Hygrostola homomunda Fletcher the male genitalia has a long and thin uncus, rounded at the apex; valves elongate and narrow; strong cucullus, club-shaped tufted with bristles, with a short neck; sacculus without clavus, presence of a sclerotized plate or spine across the upper edge of the sacculus, aedeagus short, curved, with vesica armed with rows of short stout spines. However it can be easily separated from H. homomunda with the less elongated sacculus, a sclerotized costal margin with a strong spine expansion, pointed backward, juxta large and plate-shaped, vesica with one strong cornuti, ovipositor less elongated, ostium bursae without a cup-shaped antrum.

The four Conicofrontia species collected in the field as larvae from host-plants belong to the Sesamia-like species as defined by Le Ru et al. (2006b). They are morphologically similar with ground colour pinkish buff without any markings; only C. lilomwa larvae looks different with head and thoracic shield dark brown when it is red-brown in the three other species.

Host-plant associations. We report here for the first time the host-plant associations of Conicofrontia spp. to four Andropogonae species, Cymbopogon giganteus, Cymbopogon sp., Miscanthus capensis and Saccharum officinarum. The feeding habits of Conicofrontia diamesa, C. lilomwa and C. sesamoides are similar, with the typical symptom of plant attack as death of the central tiller, often referred to as 'dead heart'. In addition, like for Acrapex spp. (Le [START_REF] Ru | Integrative taxonomy of Acrapex stem borers (Lepidoptera: Noctuidae: Apameini)[END_REF], we always found the larvae solitary in the stems. On the other hand, the feeding habits of C. bipartita is quite different with typical symptoms of plant attack as drying out of the inflorescence with second and third instar larvae found at the bottom of inflorescence, always gregarious up to 50-70 larvae. We speculate that Conicofrontia larvae typically fed on more than one stem before completing their development. The four Conicofrontia species larvae are also found to be markedly hygrophilous species inhabiting grasses along banks of streams, rivers and marshes. We suspect that the larvae disperse when they reach the fourth instar. No pupae were found in stems, and therefore borers probably pupate in the soil.

Our results suggest restricted distributions and host-plant associations of the four Conicofrontia species. Despite extensive surveys in more than 16 sub-Saharan countries we did not collect any Conicofrontia specimens in any other country than South Africa and Tanzania.

FIGURE 1 .

 1 FIGURE 1. Adults of Conicofrontia species (C. bipartita and C. diamesa). Scale bar = 10 mm.Conicofrontia bipartita: 1a-male upper side, 1b-male under side, 1c-female upper side, 1d-female under side. Conicofrontia diamesa: 1e-male upper side, 1f-male under side, 1g-female upper side, 1h-female under side.

FIGURE 2 .

 2 FIGURE 2. Adults of Conicofrontia species (C. lilomwa and C. sesamoides). Scale bar = 10 mm.Conicofrontia lilomwa: 2a-male upper side, 2b-male under side, 2c-female upper side, 2d-female under side. Conicofrontia sesamoides: 2e-male upper side, 2f-male under side, 2g-female upper side, 2h-female under side.

FIGURE 4 .

 4 FIGURE 4. Last instar larvae of Conicofrontia species. Scale bar = 10 mm. Conicofrontia bipartita: 4a; Conicofrontia diamesa: 4b; Conicofrontia lilomwa: 4c; Conicofrontia sesamoides: 4d.

  257.Conicofrontia diamesa(Hampson):[START_REF] Tams | A revision of the African species of Sesamia Guenée and related genera (Agrotidae-Lepidoptera)[END_REF]652. Material examined. Holotype (male) of Arenostola diamesa: Republic of South Africa: Zululand, Eshowe, xii 1916, Agrotidae genitalia slide 1292, E.E. Platt Coll. [BMNH]. Other material: Republic of South Africa: 3♂, 4♀, Eston, 29°55.102'S, 30°37.222'E, 673m a.s.l., 02.ii.2009, ex larva (in stem of Cymbopogon sp.) [2♂ gen. prep LERU Bruno/G300-G643, 2♀ gen. prep LERU Bruno/G55-G301] (B. Le Ru, leg.) [MNHN].

FIGURE 6 .

 6 FIGURE 6. Distribution map of sampled Conicofrontia and Hygrostola specimens.

FIGURE 7 .

 7 FIGURE 7. Results of molecular analyses. Support of major nodes is provided by BV (only BV > 50% are shown). On the right we provide the former names of two species (sensu[START_REF] Poole | Lepidopterorum Catalogus. New Series. Fasc[END_REF]. Results of PTP analyses are figured using coloured branches. Putative molecular species clusters are indicated using transitions between blue-coloured branches to red-coloured branches.

TABLE 1 .

 1 Taxon sampling. If known, rearing information is provided (otherwise the abbreviation N/A is used). Specimens belonging to the five species of interest are listed first (names follow[START_REF] Poole | Lepidopterorum Catalogus. New Series. Fasc[END_REF] with the exception of P. bipartita

  :272 Conicofrontia sesamoides: Tams & Bowden, 1953:651 Material examined. Holotype (female) of Phragmatiphila bipartita: Republic of South Africa: E. Transvaal, White River, xii 1906, Agrotidae genitalia slide 1271, A.T. Cooke Coll., 1906-314 [BMNH]. Other material: Republic of South Africa: 1♂, 1♀, Kwazulu-Natal, Karkloof, 29°16.282'S, 30°21.381'E, 1291m asl, 08.ii.2007, ex larva (in stem of Miscanthus capensis (Nees) Andersson) [1♂ gen. prep LERU Bruno/ G504, 1♀ gen. prep LERU Bruno/304] (B. Le Ru, leg.) [MNHN].

  Le Ru, sp. nov. Cymbopogon giganteus Chiov. (B. Le Ru, leg.) [MNHN]; 5♂, same data as holotype (B. Le Ru, leg.) [MNHN]; 1♂, Iringa region, Njombe, Igosi, 09°18.443'S, 34°29.132'E, 2119m a.s.l., i.2012, ex light trap (B. Le Ru, leg.) [MNHN]; 1♂, Iringa region, Sao Hill, 08°27.421'S, 35°10.036'E, 1845m a.s.l., i.2012, ex light trap (B. Le Ru, leg.) [MNHN].

	(Figs. 2a-2d, 3c, 3g, 3k, 4b)
	Type material. Holotype (male) of Conicofrontia lilomwa: Tanzania: Iringa region, Njombe, Lilomwi,
	09°36.203'S, 35°10.875'E, 1555 m a.s.l., i.2012, ex light trap [gen. prep. LERU Bruno/G88] (B. Le Ru, leg.)
	[MNHN].
	Paratypes: Tanzania: 5♂, 5♀, Iringa region, Njombe, Iboya, 09°25.541'S, 35°03.690'E, 1664 m a.s.l.,
	iv.2014, ex light trap [1♂ gen. prep. LERU Bruno/G76, 2♀ gen. prep. LERU Bruno/G682-G707] (B. Le Ru, leg.)
	[MNHN]; 1♀, Iringa region, Njombe, Lilomwi, 09°36.203'S, 35°10.875'E, 1555m a.s.l., iii.2008, ex larvae in
	stem of

  1♂, Kwazulu-Natal, Durban, xii.1909 [Agrotidae genitalia slide 1214] (G.F. Leigh, Coll.) [BMNH]; 2♂, 2♀, Kwazulu-Natal, Karkloof River, 29°13.416'S, 30°21.456'E, 1128m a.s.l., xi.2009, ex larvae in Miscanthus capensis (B. Le Ru, leg.) [MNHN]; 1♀, Kwazulu-Natal, Karkloof Forest, 29°16.282'S, 30°21.381'E, 12913m a.s.l., xi.2009, ex larvae in M. capensis, [1♀ gen. prep. LERU Bruno/G302] (B. Le Ru, leg.) [MNHN]; 1♂, 1♀, Kwazulu-Natal, Schevers Farm, 29°10.448'S, 30°21.243'E, 1053m a.s.l., xi.2009, ex larvae in M. capensis (B. Le Ru, leg.) [MNHN]; 1♂, 1♀, Kwazulu-Natal, Oak Hotel, 29°49.182'S, 30°10.414'E, 1069m a.s.l., xi.2009, ex larvae in M. capensis [1♂ gen. prep. LERU Bruno/G306] (B. Le Ru, leg.) [MNHN]; 1♂, 1♀, Kwazulu-Natal, Minerva Game Reserve, 29°47.275'S, 30°11.172'E, 1584m a.s.l., xi.2009, ex larvae in M. capensis [1♂ gen. prep. LERU Bruno/G642, 1♀ gen. prep. LERU Bruno/G305] (B. Le Ru, leg.) [MNHN]; 1♀, Eastern Cape, Slykraal, 33°22.185'S, 26°28.585'E, 348m a.s.l., xi.2009, ex larvae in M. capensis [1♀ gen. prep. LERU Bruno/G307] (B. Le Ru, leg.) [MNHN].

  , Iringa region, Njombe, Igosi, 09°18.443'S, 34°29.132'E, 2119m a.s.l., i.2012, ex light trap [1♂ gen. prep. LERU Bruno/G90, 1♀ gen. prep. LERU Bruno/G91] (B. Le Ru, leg.) [MNHN]; 2♂, 1♀, Iringa, Dabaga, 08°01.549'S, 35°51.131'E, 1832 m a.s.l., xii.2012, ex light trap (B. Le Ru, leg.) [MNHN]; 1♀, Iringa, Sao Hill, 08°27.421'S, 35°10.036'E, 1845m a.s.l., i.2012, ex light trap [1♀, gen. prep. LERU Bruno/ G98] (B. Le Ru, leg.) [MNHN].

Acknowledgments

We thank Markku Pellinen, and two anonymous reviewers for constructive comments on a previous version of the manuscript. We thank the curators of BMNH (M. Honey), MCSN (F. Rigato) and TMSA (M. Krüger) for the permission to study and photograph the types. We also thank Y. Assefa for having shown us infestation on sugarcane fields in Republic of South Africa. Financial support was provided by the Institut de Recherche pour le Développement, by icipe, African Insect Science for Food and Health (Kenya) and by the program "Bibliothèque du Vivant" (Project Noctuid Stem Borer Biodiversity; NSBB) supported by a joint CNRS, INRA and MNHN consortium. Laboratory facilities were provided by icipe, African Insect Science for Food and Health (Kenya) and the laboratory Evolution Génomes Spéciation of the Centre National de la Recherche Scientifique in Gif/Yvette (France). The authors also thank Alexandre Dehne Garcia for his help on the CBGP HPC computational platform. All specimens were collected under appropriate collection permits from the two countries recorded and no conflicts of interest were discovered.


Bernadette Julier

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Farmer AD, Fouteau S, Franken C, Dudez Am

Gish J Goldstein

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From the model legume Medicago truncatula to alfalfa

Keywords: genomics, marker, model species, QTL, sequence

Large efforts have been devoted to Medicago truncatula genomics. They offer data and tools to identify M. truncatula genes involved in genetic variation for traits related to agronomic value in crop species. This strategy is based on the hypothesis that the same genes are involved in trait variation in both species. These tools are now used to develop alfalfa genomics in order to carry out genomic studies in the target species. Identification of genes or markers in the crop species will contribute to develop tools and theory to implement marker assisted selection in alfalfa.

Many efforts have been devoted to Medicago truncatula Gaertn. genetics and genomics since 1990. A draft sequence has been obtained after sequencing the gene-rich genome portions (euchromatin) (9). Its analysis showed that M. truncatula genome is more complex than expected from its short genome size. The genome has been shaped by a whole-genome duplication but also by numerous rearrangements and local duplications. These genome changes allowed the emergence of specific genes involved in rhizobial nodulation. The number of genes is over 62,000, and the genome contains numerous transposons, retrotransposons and transcription factors. Resequencing of a set of 26 accessions has given access to sequence polymorphism [START_REF] Branca | Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula[END_REF]. A large diversity was found, but linkage disequilibrium was shorter than expected in a selfing species. Transcriptomics data were used to construct a genechip array (https:www.affymetrix.com /products_services/arrays/specific/medicago .affx) that can be used to detect the role of genes in specific functions and organs. In parallel, accessions collected from sites of the Mediterranean Basin were made available to the scientific community in gene banks (http:/ / www1.montpellier.inra.fr/BRC-MTR/).

From M. truncatula to alfalfa: strategies

Exploiting genomic knowledge from M. truncatula for the genetic improvement of alfalfa is not simple. Detecting genes involved in the variation for breeding traits is often needed to improve breeding programs. Two non-exclusive strategies can be devised. In the first one, candidate genes are detected in M. truncatula and are then tested for their effect in crop species (Figure 1). In the second one, the genomics of alfalfa is developed by taking into account the knowledge from M. truncatula genomics, and used to detect genes or markers involved in traits. These genes or markers become available to be incorporated into breeding programs (Figure 1).

Identification of genes in M. truncatula

In the first strategy, the QTL detection in mapping populations of M. truncatula, followed by a fine mapping step, gives access to a small genomic region. Using an in silico analysis of the genes annotated in the QTL region and a literature review of the genes known to be involved in the trait under study, a list of candidate genes can be established. This method was successfully used for flowering date. One of the candidate genes, a Constans-like gene, contained sequence polymorphism and showed differential expression between the two parents (7). Analysis of mutants with altered phenotypes (forward genetics), or phenotypic analysis of genotypes showing differential gene expression (reverse genetics) are other ways to detect candidate genes. Association genetics in M. truncatula could also help to identify genes or genomic regions involved in traits. The efficiency of association genetics will depend on the linkage disequilibrium (LD) of the population under study, which can be long in this selfing species.

RESEARCH

Once a gene has been identified in the model species, it remains to verify the presence of a homeologue of it in alfalfa which has an effect on the same trait. This demonstration can be achieved and exploited by different methods, such as transgenesis, introgression through backcross, or association genetics (Fig. 1). With transgenesis, the modification of expression of an allele in alfalfa is supposed to confer a new trait variation (8), eventually becoming available in a transgenic variety. Introgression through backcross has the same objective, but the allele originates from alfalfa. With association study, the demonstration results in the identification of alfalfa allele(s) that confer valuable variation, as found for a Constans-like gene [START_REF] Herrmann | Association of a CONSTANS-LIKE gene to flowering and height in autotetraploid alfalfa[END_REF]. These alleles could then be used in a breeding program assisted by molecular markers, to increase their frequency in the synthetic varieties.

Development of alfalfa genomics

The second strategy implies the study of alfalfa genomics by DNA or cDNA sequencing using the Next Generation Sequencing (NGS) technologies (Fig. 1). Read assembly is complex because each individual possibly carries 4 different copies of each gene, in addition to the presence of gene families or repeated elements. The assembly of alfalfa sequences can be considered as a re-sequencing, with M. truncatula sequence acting as a reference. However, extensive sequence evolution occurred in the two species, leading to large differences in non-coding sequences between M. truncatula and alfalfa. Alfalfa sequencing is currently a scientific challenge that is addressed by a few groups in Europe and the USA. In the meanwhile, genetic mapping and QTL detection are feasible. Theories and software (TetraploidMap, http://www.bioss.ac.uk/knowledge/tetraplo idmap/) were developed in autotetraploid species. Microsatellite markers were developed in alfalfa which mostly originated from EST of M. truncatula (6). These codominant markers with additional dominant markers such as AFLP or DArT (3) or HRM (4) markers are numerous enough to build maps of any mapping population. The comparison of M. truncatula and alfalfa maps proved that both macrosynteny and micro-synteny between the two species are high. Due to autotetraploidy, the precision of QTL position is lower in alfalfa than in a diploid species, so the subsequent use of QTL into breeding is still limited to the genetic background of the mapping population. Fine QTL mapping has not been reported in alfalfa, probably because it would require very large mapping populations and in silico analysis of genomics regions is not feasible yet. However, this in silico analysis, by using the same anchor markers in alfalfa and M. truncatula, can be carried out in the model species. On the whole, gene or marker detection directly performed in alfalfa (through bulk segregant analysis (BSA) or genomic selection) is scarce up to now, but the increasing availability of high throughput markers could change this situation in the future. These markers will now be mainly developed in alfalfa from sequencing and resequencing programs. However, any analysis of their function, position on maps, sequence polymorphism of their genomic regions will benefit from genomic resources in M. truncatula.

Perspectives

Current development of alfalfa genomics, now possible because of extensive knowledge in M. truncatula, offers new perspective to directly detect genes or markers in the crop species for agronomics traits. All limitations to genetic analysis in alfalfa are not yet overcome and more specifically, polyploidy and heterozygosity will lastingly reduce their efficiency. So M. truncatula is still a reference, mainly for all the -omics data, but also for the identification of genes involved in agronomic traits. ■

Figure 1 .

 1 Figure 1. Strategies to transfer genomics information from M. truncatula to the breeding program of a legume species; several methodologies (boxes) are proposed to achieve each step