A new species of Mengenilla (Insecta, Strepsiptera) from Tunisia

Abstract A new species of Mengenilla Hofeneder, 1910 (Strepsiptera, Mengenillidae) from southern Tunisia is described. Mengenilla moldrzyki sp. n. can be distinguished from congeners by a slightly emarginated posterodorsal margin of the head, compound eyes with a light tan dorsal part, mandibles with a narrow distal part, and a v-shaped pronotum. With the description of Mengenilla moldrzyki sp. n., eleven valid species of Mengenilla are currently recognised. Mengenilla moldrzyki sp. n. is the third species of the genus with known females and female puparia. First instar larvae, endoparasitic larval stages, the male puparium and the host are unknown. The new species is also the first strepsipteran with a fully sequenced genome.


Introduction
Mengenillidae is a basal group of Strepsiptera. The recently described Bahiaxenos relictus Bravo, Pohl, Silva-Neto and Beutel, 2009 (Bahiaxenidae) from Brazil is the sistergroup of all other strepsipteran families, and Mengenillidae represents the second branch within the order, i.e. the sistergroup of all families with pterygote hosts (Stylopidia) (Pohl andBeutel 2005, Bravo et al. 2009). Plesiomorphic features including free-living females, comparatively broad mandibular bases, and the absence of specialized hairy soles on the tarsomeres of males characterize the group. Recently, the monophyly of Mengenillidae was challenged. If only characters of males are analysed the mengenillid genera Eoxenos, Congoxenos, and Mengenilla split successively from the strepsipteran phylogenetic backbone after Bahiaxenos , Bravo et al. 2009, Hünefeld et al. 2011. The clarification of this issue will require detailed morphological information on males, females and larvae -information that is presently still very fragmentary. The monophyly of Mengenilla is well established and supported by the following apomorphies of males: reduced primary and secondary mandibular joint, immobilized maxilla, and a completely undivided labium . Further diagnostic features of the males are six-segmented antennae, flabella on antennomeres 3-5, five-segmented tarsi with well developed claws, a prominent and elongated abdominal segment X, and a nearly straight penis with a pointed apex (Kinzelbach 1970, 1978, Cook 2007.
A total of 19 species have been described in the genus Mengenilla, 10 of which are currently recognised as valid (Cook 2007). The genus is restricted to the Old World and occurs in xerotherm, semi-arid and arid areas, with the exception of M. orientalis Kifune and Hirashima, 1980 from Sri Lanka and M. leucomma Cook, 2007 from Madagascar (Kinzelbach 1979, Cook, 2007, Bravo et al. 2009). Mengenilla chobauti Hofeneder, 1910 andM. parvula Silvestri, 1941 occur in the Mediterranean region. While M. parvula is restricted to Sicily, M. chobauti is recorded from North Africa, Spain, Portugal, Crete, Malta, Italy, including Sicily and Sardinia and has the widest distribution of all described Mengenilla species (Silvestri 1941, 1943, Kinzelbach 1978. Nearly all other species are only known from their type locality. Figure 1 gives an overview of the described species of the genus and their distribution. Males are known for all currently described species of Mengenilla, but females, immature stages and hosts only for M. chobauti and M. parvula. The presently known hosts are lepismatid Zygentoma: Ctenolepisma ciliata (Dufour, 1831) for M. chobauti, and C. michaelseni Escherich, 1905for M. parvula, respectively (Silvestri 1943. In this contribution, we describe a new species of Mengenilla from southern Tunisia. In October 1999, the first author (HP) collected more than 260 male specimens at a light trap in the Tunisian Sahara. Despite an intensive search, females were neither found by that time nor during a second collecting trip in 2010. In 2005, Uwe Moldrzyk (Berlin) collected a single female within its puparium. Several studies have already been published on males of this species without a formal description (referred to as "Mengenilla sp., undescribed species Tunisia"). They cover the head morphology (Beutel and Pohl 2006), effects of miniaturization , the male postabdomen, and genital structures Beutel 2005, Hünefeld et al. 2011). Finally, the complete genome of this species has been sequenced, which is also the first published complete genome of a twisted-wing parasite (Niehuis et al. in press). Mengenilla moldrzyki sp. n. is consequently the best investigated strepsipteran species thus far.

Methods
The morphological terminology for the head is based on Beutel and Pohl (2006) and for the thorax and abdomen on Kinzelbach (1971Kinzelbach ( , 1978, except for the term "aedeagus", which is replaced by the term "penis" as suggested by Hünefeld et al. (2011). Species descriptions are based on a designated holotype but all available specimens were taken into account in order to assess the intraspecific variation.  Figure 1. Distribution of the genus Mengenilla plotted on the Köppen-Geiger climate type map (map modified after Peel et al. (2007)). Type localities with open symbols. Grey dots: Mengenilla sp. mentioned by Hofeneder (1928) and de Peyerimhoff (1919). Description of the climate symbols (only where Mengenilla occurs): Aw Tropical, savannah; BWh Arid, desert, hot; BWk Arid, desert, cold; BSh Arid, steppe, hot; BSk Arid, steppe, cold; Csa Temperate, dry summer, hot summer; Csb Temperate, dry summer, warm summer. For a detailed explanation of the map and symbols see Peel et al. (2007).
Photographs of critical point dried specimens were taken with a Nikon D 90 digital SLR equipped with a 40 mm and with a 63 mm Zeiss Luminar macro lense, plus an adjustable extension bellows. The specimens were illuminated by two flashlights fitted with a transparent cylinder for even and soft light. Helicon Focus Mac Pro X64 was used to combine a stack of several partially focused images. For scanning electron microscopy images (SEM) and macro photography, specimens were dehydrated using increasing steps of ethanol up to 100% and dried at the critical point (Emitech K850 critical point dryer). For SEM, specimens were subsequently sputter-coated (Emitech K500) and examined on a Philips XL30 ESEM using a rotatable specimen holder (Pohl 2010).
To assess intraspecific variation, 74 male specimens of Mengenilla moldrzyki sp. n. were prepared on slides and embedded in Euparal (Chroma, Münster, Germany). To avoid shrink-artefacts, specimens were dehydrated using increased steps of ethanol up to 96% prior to preparation, followed by several steps of Euparal Essenz in increasing concentration (diluted with 96% ethanol) up to 80% and then finally embedded in Euparal. An antenna, a maxilla, legs, the hind wing of one side, and the penis were prepared and each covered separately with a coverslip. Measurements of the 74 prepared males were performed using an Olympus SZ 40 stereomicroscope and an Olympus BX 50 microscope with a calibrated ocular micrometer. Measurements of the female and of the female puparium were performed using SEM micrographs and macro photographs. Definitions of the measurements are illustrated in figure 4 and in figure 5. Morphological features were illustrated using a 10×10 ocular grid on either an Olympus SZ 40 stereo microscope or an Olympus BX 50 microscope. Drawings of features of the females are based on SEM micrographs. The colouration of different body parts of the specimens are specified after the List of colours (Wikipedia).
To corroborate our assumption that the males and the females described here indeed belong to the same species, i.e., Mengenilla moldrzyki sp. n., we compared a 741-bp long section of their mitochondrial gene COI -an established barcoding gene. DNA was extracted from a small part of the abdomen of the adult female using the QIAGEN DNeasy Blood & Tissue Kit and following the protocol for insects (QIAGEN GmbH, Hilden, Germany). The COI marker region was PCR-amplified with the oligonucleotide primers 5'-TAG GGG TTA GAT CAG GTT GA-3' and 5'-AGG ACA TAG TGG AAA TGT GC-3'. The oligonucleotide primers had been specifically designed for this purpose with the software Primer3 (Rozen and Skaletsky 2000), using the COI sequence from the M. moldrzyki genome project (Niehuis et al. in press) as reference and template. Note that the M. moldrzyki genome has been sequenced using the DNA from males (Niehuis et al. in press). The PCR was conducted in a 20 μl volume consisting of 0.5x QIAGEN Q-Solution, 1x QIAGEN Multiplex PCR Master Mix (QIAGEN GmbH, Hilden, Germany), 0.8 μM of each oligonucleotide primer, and ~ 50 ng DNA. The PCR temperature profile started with an initial denaturation and QIAGEN Hot-StarTaq DNA polymerase activation step at 95° C for 15 min., followed by 35 cycles of 95° C for 1 min., 49° C for 1 min., and 72° C for 1 min, followed by 10 min. at 72° C. The PCR product was purified with the QIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany) and send to Macrogen Inc. (Amsterdam, Netherlands) for direct sequencing with the above oligonucleotide primers. Forward and reverse DNA strands were assembled to contigs, trimmed (to exclude the binding sites of the oligonucleotide primers), and aligned to the COI reference sequence from the M. moldrzyki genome project (Niehuis et al. in press) with GENEIOUS PRO 5.5.3 (Drummond et al. 2010). The assembled COI sequence has been deposited in the European Nucleotide Archive (ENA) und is available under the accession number HE610110.
The information for the specimens is given in a standard manner, i.e., locality, geographic coordinates, elevation, date of collection (month indicated in lower case Roman numerals), habitat information, collector, depository, and preparation. Male (♂) and female (♀) symbols indicate the sex.
The specimens referred to below along with the abbreviations used in the text are Diagnosis. Six-segmented antennae with flabella on antennomeres 3-5, immobilized maxillae, a completely undivided labium, and five-segmented tarsi with well developed claws are generic features of the genus Mengenilla verified in the new species.
The males are distinguished from congeners as follows (M. marikovskii Medvedev, 1970 from south-eastern Kazakhstan is excluded from the key, because the description and illustrations are too superficial. In contrast to the statement of Medvedev (1970), the type series of that species is not deposited in the Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia and could not be re-examined.): 1 Antenna relatively short (less than 2 times the head length The female of M. moldrzyki sp. n. is distinguished from M. chobauti and M. parvula by the much more slender distal part of its mandible, and from M. parvula additionally by its longer scapus. The female puparium is distinguished from that of M. chobauti and M. parvula by the complete absence of cuticular thorns, a rounded anterior prothoracic margin with rounded anterolateral edges, and a tapering caudal margin of the abdomen (Figs 12, 13).
Description of the male (Figs 2-6). Measurements (male holotype, followed by minimum, maximum of paratypes, and mean values of all measured specimens in parentheses, critical point dried specimens and specimens in ethanol not measured, in μm): 1. total length 4,000 (3,450-4,785, avg. 4,182)   Head capsule (Figs 2, 3A, 3C): dorsal side cream-coloured, lateral part brownish, subgenal region distinctly darkened at mandibular base and postoccipital ridge. Subprognathous, slightly to distinctly inclined; broader than long and strongly narrowed immediately posterior to compound eyes, not retracted into prothorax; laterocervicalia absent; posterodorsal margin of head emarginated; dorsal side densely covered with microtrichia; some short setae present on vertex, but absent from frons; area posterior to compound eyes and ventral side of head capsule glabrous and very smooth; ocelli absent; compound eye very large and extending to ventral side of head, composed of 44-77 large ommatidia (avg. 65); mediodorsal part creamy-white, lateral and ventral part seal brown; dorsal and lateral ommatida widely separated and intervals densely covered with microtrichia; ventral ommatidia slightly larger and closely adjacent; deep lyriform frontal impression present on dorsal side of head and longitudinal bulge laterally; bulges camel-coloured, anteriorly forming antennal insertion; transverse frontoclypeal strengthening ridge absent; anterior clypeofrons slightly emarginated, laterally separated from genal region by dark narrow zone ending posteriorly at antennal insertion; median part parallel-sided, brighter and more densely covered with microtrichia than lateral region; cranial part inflected, separated from frontal mandibular base and mouthfield sclerite by membranous area with distinct median brownish stripe extending laterad; subgenal area and gena seal brown; ventral head closed by lateral postgenal area and median undivided labial plate between maxilla and postgenal region; mouth opening transversely oval.
Abdomen (Figs 4, 5D, 6C): tergites less strongly sclerotised than sternites, brown; tergite and sternite of segment I reduced; tergite II partly covered by metapostnotum; tergites II-VIII, rectangular, increasing in width from segments II-VIII; pleural membrane of segments I-VIII wide, camel coloured; spiracles present on segments I-VII; shape of sternites II-VIII similar to corresponding tergites but distinctly broader, shovel-shaped, brown; segment IX strongly sclerotised, distinctly narrower than segment VIII, with caudally elongated subgenital plate; segment X tube-like, extending above tip of subgenital plate; penis curved, with bulbous proximal part; acumen thin, tapering towards apex. Description of the female (Figs 7-10). Head capsule (Figs 7-9, 10A-10C): uniformly tan, with the exception of articulatory membranes of antenna and membranised ventral head areas. Relatively small, distinctly broader than long, and cuneiform in lateral aspect; orthognathous, with the posterior part distinctly retracted into the prothorax; laterocervicalia absent; dorsal side smooth, without vestiture of microtrichia; short setae present on anterior clypeal region, few setae present above antennal insertion and area posterior to compound eyes; ocelli absent; compound eye small, composed of 11-12 large, equally sized and closely adjacent ommatidia; microtrichia between ommatidia absent; central part of compound eye grey, peripheral ommatidia tan; circumocular ridge slightly darker; frontal impression on dorsal side of head absent; ventral side of head closed by membranised reduced labium and possibly cervical membrane; mouthfied sclerite small, oval, reaching posterior margin of maxilla; mouth opening transverse, oval.
Abdomen (Fig. 7): ivory-coloured and very weakly sclerotised; abdominal segments strongly arched dorsally, ± flattened ventrally; spiracles at lower third of segments I-VII; fissure-shaped birth opening present on posterior border of segment VII.
COI sequence: 100% identical between the female and the sequenced males. Description of the female puparium (Fig. 11). Measurements: total length 5,700, maximum width 2,800, maximum height 1,600, length of legs without claws 300.
Head missing (already shed); dorsal side of puparium strongly arched, ventral side flattened; anterior margin of prothorax rounded and forming distinct bulge with rounded anterolateral edge; anterior third of metathorax slightly constricted; legs very short, inserted laterally, with thread-like claws; caudal margin of abdomen tapering; spiracles present at abdominal segments I-VII.

Discussion
The type locality of M. chobauti is Ain Sefra in Algeria. All other species of Mengenilla described from northern Africa (i.e., M. theryi (Hofeneder, 1926), M. mauretanica (Hofeneder, 1928) (both from Morocco), M. santchii (Pierce, 1918) (from Tunisia)) are today considered as junior subjective synonyms of M. chobauti (Kinzelbach 1970, Cook 2007. We re-examined all available material (including all types) of these species, and encountered a considerable amount of variation, especially in the length of antennomere 6, the length of the flabella, and the shape of the maxilla and penis. As pointed out by Cook (2007), it cannot not be excluded that more than one species occurs in this region. However, all synonymised North African species, including M. santchii from Tunisia, differ significantly from M. moldrzyki by the broad distal part of their mandible.

Recommendations for future species descriptions
Important diagnostic features of males of species of the genus Mengenilla are the shape of the posterodorsal margin of head, the dorsomedian frontal impression, the total length of the antennae, the tips of the flabella and antennomere 6, the shape of the distal part of the mandibles, the proximal part of the maxilla, the insertion of the maxillary palp, the shape of the pronotum, and the length of the metapostscutellum. In contrast, the total length, the proportions of the maxilla and the maxillary palp as well as the shape of the penis are very variable and only partly suitable for species identification (compare Fig. 6). Kinzelbach (1979) and Cook (2007) refer to the different patterns of microtrichia on the mandibles. This feature seems to be suitable for diagnosis, but the vestiture varies greatly on the frontal, lateral and ventral mandibular areas (Fig. 3). It is not clear from previous descriptions and illustrations from which perspective the isolated mandibles are shown. This greatly reduces the value of the presented information (see Cook 2007: Fig 5). If more than one specimen is available, standard views of the head in dorsal, frontal, and lateral view should be given (and documented by SEM if possible) in addition to drawings of the antenna, mandible, maxilla, dorsal view of the whole insect, the legs, the hind wing, and the penis. With the use of the SEM specimen holder developed by Pohl (2010), a single specimen can be examined from all sides. Differences in colouration also appear suitable for diagnosis but have rarely been used in the literature so far. Unfortunately, older specimens treated with potassium hydroxide solution and embedded in Canada balsam on slides are no longer useful for this purpose. To document the colouration, light micrographs of the head and thorax in dorsal view and of the head in frontal view should be given. Only with welldocumented descriptions, a reliable identification of other conspecific individuals is possible without comparing it to the type specimens.