Hidden Diversity—A New Speciose Gall Midge Genus (Diptera: Cecidomyiidae) Associated with Succulent Aizoaceae in South Africa

Simple Summary Succulent Aizoaceae (often called “mesembs” or ice plants) form a dominant component of the Succulent Karoo in southern Africa, constituting one of the most species-rich families within the Greater Cape Floristic Region (GCFR). Despite the diversity and abundance of these plants, the diversity of insects specialized on them has never been surveyed methodically prior to this study. In a three-year study of the galling insects associated with succulent Aizoaceae in South Africa, we found that they support a rich community of gall midges (Diptera: Cecidomyiidae), virtually all of which are new to science. This is not surprising, given that knowledge of the Afrotropical fauna of gall midges is scarce, with most species described in the 1900s. Here, we describe the new genus Ruschiola with ten species from succulent Aizoaceae in Namaqualand, the Knersvlakte and the Cedarberg regions of South Africa based on morphological, molecular and life history data. The genetic data were particularly important in this study for differentiating taxa, given that Ruschiola species are very similar morphologically. Members of this genus develop in leaf galls or in plant tissues without visible gall formation, and are highly host specific. Abstract Aizoaceae (Caryophyllales) constitute one of the major floral components of the unique Greater Cape Floristic Region (GCFR), with more than 1700 species and 70% endemism. Within succulent Aizoaceae, the subfamily Ruschioideae is the most speciose and rapidly diversifying clade, offering potential niches for the diversification of specialized herbivorous insects. Nevertheless, insect diversity on these plants has not been studied to date, and knowledge of gall-inducing insects in the Afrotropics is generally scarce. Our recent observations indicate that succulent Aizoaceae in the GCFR support a rich and largely unstudied community of gall midges (Diptera: Cecidomyiidae). Here, we provide a first report of their diversity with a description of a new genus, Ruschiola Dorchin, and ten new species, based on morphological and molecular analyses of material collected during a three-year targeted survey across major GCFR vegetation types. A high degree of morphological uniformity in Ruschiola suggests recent diversification and necessitated the use of molecular data and laboratory rearing from host plants to verify species boundaries and host ranges.

for many gall midges and that the distribution patterns of galling insects in general are driven primarily by the abundance of gall midges [22,23]. Our findings also support the hypothesis that plant diversity, and particularly the presence of species-rich plant families, provides the opportunity for the diversification of gall inducers [17,22,24,25].
Here, we describe the new cecidomyiid genus Ruschiola Dorchin and ten new species within it from succulent Aizoaceae in South Africa. The newly described genus belongs to the tribe Lasiopterini, the greatest diversity of which occurs in deserts and saline habitats of the eastern hemisphere, primarily on plants of the family Chenopodiaceae [9,26]. The new genus is morphologically rather uniform, possibly due to relatively recent and rapid radiation, thus requiring genetic data to confirm species boundaries and host associations. It appears to be restricted to the subfamilies Mesembryanthemoideae and Ruschioideae of the Aizoaceae (commonly known as "mesembs" or ice plants), with most of the diversity associated with the core ruschioids (tribes Ruschieae and Drosanthemeae). All known species in this genus develop in the leaves of their host plants, with or without obvious or visible gall formation.

Insect Collecting, Rearing and Preservation
The field collection of galls and other plant material was conducted over three years, from 2017 to 2019, at multiple sites in the Western and Northern Cape provinces of South Africa, including the three major biodiversity hotspots for succulent Aizoaceae, i.e., the Richtersveld, Knersvlakte and Little Karoo, as well as the major vegetation types within the GCFR [2,4]. Collecting was performed mainly during the austral winter and spring (July-September), which constitutes the peak growth season for succulent Aizoaceae in South Africa, and several sampling excursions were also conducted at other times of the year (January-April). The sampling included multiple field trips to sites in Namaqualand, the Knersvlakte, West Coast, Overberg and Little Karoo ( Figure 1).   Due to severe drought conditions that occurred during most of the study period, only the southernmost part of the Richtersveld (Vyftienmyl se Berg Inselberg in southern Richtersveld) was visited. More than 60 sites were visited repeatedly to gain insight into the abundance and phenology of the gall midges and to sample putative host plants across the major clades of succulent Aizoaceae [27,28]. Vouchers of host plants were prepared in a plant press to aid identification, oven dried as necessary, and are deposited in the Bolus Herbarium (BOL), Department of Biological Sciences, University of Cape Town.
Galls and other plant material were collected in mesh bags in the field and placed in ventilated rearing cages on the same day to enable the rearing of adult gall midges. A subset of each gall type was dissected under a stereo microscope in the laboratory to document the developmental stage of the galls and to obtain the larvae and pupae of the gall midges for morphological study. Adults, larvae and pupal exuviae were preserved in 70% ethanol and mounted on permanent microscope slides in Euparal according to the method detailed in Gagné [29]. Pupae were preserved in 70% ethanol and then prepared for scanning electron microscope imaging via chemical drying and gold sputtering. A few adults of some species were also double mounted on micro pins to preserve their color pattern, created by the thick cover of black and white scales on their body. Samples of each morpho-species (mostly adults, occasionally pupae or larvae) were preserved in 99% ethanol for genetic analysis.

Taxonomy
Morphological structures of the gall midges were studied and illustrated with the aid of a drawing tube mounted on a Leica DM1000 compound microscope (Leica Microsystems, Wetzlar, Germany). Some structures were photographed with a Leica DFC495 camera mounted on a Leica M205 C stereo microscope (Leica Microsystems, Wetzlar, Germany). Pupae were studied on a ThermoScientific Phenom XL scanning electron microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA). Terminology for adult morphology follows McAlpine [30]; terminology for wing venation follows Cumming and Wood [31], and terminology for immature morphology follows Gagné [32]. Specific terminology for the lasiopterine ovipositor follows Dorchin [33].
Holotypes are deposited in the Iziko South African Museum, Cape Town, South Africa (SAMC). Paratypes are deposited in SAMC and in the Steinhardt Museum of Natural History, Tel Aviv University, Israel (SMNHTAU), except for representative paratypes, which are deposited at the Zoologisches Forschungsinstitut und Museum Alexander Koenig, Bonn, Germany (ZFMK), and the Essig Museum of Entomology, Berkeley, CA, USA (EMEUC), as detailed in the species descriptions below. All specimens are mounted on permanent microscope slides in Euparal, except for pupae, which are mounted on SEM stubs. All localities mentioned in the examined sections of the species descriptions are in South Africa.

Molecular Methods
The dataset analyzed included 47 individuals of 10 putative species within the newly described genus and seven outgroup taxa from other Lasiopterini genera. Our aim with this analysis was to test the validity of the new genus and species and establish the host ranges of species that appeared to have multiple hosts. GenBank accession numbers for the samples included here are provided in Table 1. Genomic DNA was extracted from whole individual adults or immature midges using the Genaid Genomic DNA Mini Kit (Genaid, Taipei, Taiwan) or the Qiagen DNeasy Kit (Qiagen, Valencia, CA, USA). A fragment of approximately 680 base pairs of the mitochondrial COI gene was PCR amplified with the primers LCO1490 and HCO2198 [34]. PCR conditions consisted of 10 min denaturation at 95 • C followed by 35 cycles of: 30 s denaturation at 95 • C, 1 min annealing at 50 • C, 1 min extension at 72 • C and final extension at 72 • C for 4 min. PCR reactions were performed in a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) and products were purified using an EXO-SAP enzymatic cleanup (Thermo Scientific, Vilnius, Lithuania). Sequencing in both directions was performed on an ABI PRISM 3730xl DNA analyzer at Hy Laboratories, Rehovot, Israel. Forward and reverse strands were combined using Sequencer 4.7 (Gene Codes) and all sequences were translated into amino acids to check that no stop codons were present.

Phylogenetic Analysis
The loci were aligned using MAFFT [35] and a maximum likelihood phylogeny constructed in RAXML v8.2.12 [36] on the CIPRES portal [37]. Seven outgroups from other Lasiopterini genera were selected based on Dorchin et al. [38] and the phylogeny was rooted on Asteromyia carbonifera, belonging to the sister tribe, Alycaulini. Node support was evaluated using the rapid bootstrapping procedure implemented in RAXML. A GTR model of nucleotide substitution was applied to each codon position independently. Uncorrected pairwise sequence divergences were calculated using Paup* [39].

Molecular Results
Our molecular analysis verified the monophyly of Ruschiola and of all ten species described here (bootstrap values of 87-100%) ( Figure 2). Mean intraspecific sequence divergence was 0.0-1.1%, and mean interspecific divergence among Ruschiola species was 2.6-10.2% (Table 2). Sequence divergence between Ruschiola and the Lasiopterini outgroups used here averaged at 10.4%. Ruschiola attenuata from Mesembryanthemum splendens was retrieved as sister to all other species included here that develop in Ruschioideae host plants, paralleling the sister position of Mesembryanthemoideae relative to the Ruschioideae within Aizoaceae [5,27]. Ruschiola attenuata is also the only species that is relatively easily to distinguish from other Ruschiola species based on adult morphology. The high degree of morphological uniformity among other Ruschiola species makes it difficult to distinguish between them without genetic data and detailed information on host-plant associations, which may be indicative of recent diversification. distinguish between them without genetic data and detailed information on host-plant associations, which may be indicative of recent diversification.

Ruschiola Dorchin, New Genus
Type species: Ruschiola succulenta Dorchin and van Munster Diagnosis: Ruschiola is a striking, morphologically uniform genus of medium to large-sized lasiopterines. The adult body is densely covered by black and white scales, whereby abdominal tergites are almost completely black, with only a thin transverse line

Ruschiola Dorchin, New Genus
Type species: Ruschiola succulenta Dorchin and van Munster Diagnosis: Ruschiola is a striking, morphologically uniform genus of medium to largesized lasiopterines. The adult body is densely covered by black and white scales, whereby abdominal tergites are almost completely black, with only a thin transverse line of white scales along the posterior margin ( Figure 3A). This contrasts with the typical color pattern of most lasiopterine genera from Chenopodiaceae (e.g., Baldratia Kieffer, Stefaniola Kieffer, Careopalpis Marikovskij), in which scales on the abdominal tergites form two or three black triangles on a white background (or occasionally vice versa). The number of antennal flagellomeres is irregular within a species, as is also usual for Lasioptera Meigen, Baldratia and Stefaniola, and contrary to the invariable number of 10 flagellomeres in Careopalpis and Suaediola Dorchin. Palpi are one or two segmented and their shape may vary considerably within the same species and individual (e.g., Figure 4B). Vein R 4+5 of the wing is notably longer than in most other lasiopterine genera, joining C at about three quarters of the wing length rather than around its mid-length ( Figure 3C). The ovipositor is much shorter than that of Lasioptera and Ozirhincus Rondani, but longer than that of most lasiopterine genera from Chenopodiaceae. It is very uniform within the genus, with a well-developed, heavily sclerotized lateral plate that does not form a clear aculeus posteriorly, contrary to the distinct, ventrally curved aculeus in Baldratia and Careopalpis, straight aculeus in Stefaniella Kieffer, or dorsally curved aculeus in Stefaniola, Izeniola Fedotova and Suaediola. Pupae have well-developed antennal horns but no facial horns, and larvae have a bidentate sternal spatula, conspicuous and long shafted in some species to vestigial and barely visible in others.
Insects 2022, 13, x FOR PEER REVIEW 9 of 36 of white scales along the posterior margin ( Figure 3A). This contrasts with the typical color pattern of most lasiopterine genera from Chenopodiaceae (e.g., Baldratia Kieffer, Stefaniola Kieffer, Careopalpis Marikovskij), in which scales on the abdominal tergites form two or three black triangles on a white background (or occasionally vice versa). The number of antennal flagellomeres is irregular within a species, as is also usual for Lasioptera Meigen, Baldratia and Stefaniola, and contrary to the invariable number of 10 flagellomeres in Careopalpis and Suaediola Dorchin. Palpi are one or two segmented and their shape may vary considerably within the same species and individual (e.g., Figure 4B). Vein R4+5 of the wing is notably longer than in most other lasiopterine genera, joining C at about three quarters of the wing length rather than around its mid-length ( Figure 3C). The ovipositor is much shorter than that of Lasioptera and Ozirhincus Rondani, but longer than that of most lasiopterine genera from Chenopodiaceae. It is very uniform within the genus, with a welldeveloped, heavily sclerotized lateral plate that does not form a clear aculeus posteriorly, contrary to the distinct, ventrally curved aculeus in Baldratia and Careopalpis, straight aculeus in Stefaniella Kieffer, or dorsally curved aculeus in Stefaniola, Izeniola Fedotova and Suaediola. Pupae have well-developed antennal horns but no facial horns, and larvae have a bidentate sternal spatula, conspicuous and long shafted in some species to vestigial and barely visible in others.   The genus currently includes the ten species described here, but many more are likely to be found as the full diversity of Aizoaceae host plants in South Africa and Namibia is yet to be explored. All known Ruschiola species develop in conspicuous leaf galls or in leaf tissues without visible external deformation. Many plants that host Ruschiola species are also inhabited by Asphondylia Loew species, which gall buds or flowers rather than leaves, and will be dealt with elsewhere. The evolutionary relationships between Ruschiola and other genera in the Lasiopterini are unclear at present and warrant further study.
Adult. Head ( Figure 3B): Eye facets round; gap between eyes on vertex 0-2 facets wide. Antenna: scape cylindrical, pedicel globose, both densely covered by long, dark scales; flagellomeres 10-18, barrel shaped to nearly quadrate ( Figure 3B), 1-2 times as long as wide, apical flagellomere either shorter or longer than preceding flagellomeres, The genus currently includes the ten species described here, but many more are likely to be found as the full diversity of Aizoaceae host plants in South Africa and Namibia is yet to be explored. All known Ruschiola species develop in conspicuous leaf galls or in leaf tissues without visible external deformation. Many plants that host Ruschiola species are also inhabited by Asphondylia Loew species, which gall buds or flowers rather than leaves, and will be dealt with elsewhere. The evolutionary relationships between Ruschiola and other genera in the Lasiopterini are unclear at present and warrant further study.
Adult. Head ( Figure 3B): Eye facets round; gap between eyes on vertex 0-2 facets wide. Antenna: scape cylindrical, pedicel globose, both densely covered by long, dark scales; flagellomeres 10-18, barrel shaped to nearly quadrate ( Figure 3B), 1-2 times as long as wide, apical flagellomere either shorter or longer than preceding flagellomeres, rounded or tapered apically, often consisting of two merged flagellomeres; each flagellomere with two whorls of appressed circumfilla with 1-2 longitudinal connections and two more-or-less ordered whorls of strong setae; setae of distal whorl strongly curved at base, stronger, longer and originating from larger sockets than setae of proximal whorl; flagellomeres evenly setulose elsewhere. Frontoclypeal membrane with many long hair-like setae and scales. Palpi 1-2 segmented, shape may vary within species and individual. Labella diminutive to well developed.
Thorax: Wing ( Figure 3C) densely covered by delicate setulae, posterior margin fringed by long, delicate hair-like setae; C broken beyond junction with R 4+5 ; R 4+5 reaching C at about three quarters of wing length; C and R 4+5 thick, densely covered by black scales to meeting point; M present as fold; CuA 1 and CuA 2 form a fork. Legs: Tarsal claws toothed on all legs, tooth curved close to base; empodia as long as or much longer than bend in claws; pulvilli about half as long as claws.
Female abdomen ( Figure 3D): Densely covered by scales; tergites mostly black, except for thin stripe of white scales along posterior margin. Tergites 1-7 rectangular, with anterior pair of sensory setae, posterior row of strong setae, and evenly distributed scales; tergite 8 with considerably reduced pigmentation or not differentiated from surrounding membrane, without vestiture other than anterior pair of sensory setae. Sternites 2-6 rectangular, without anterior sensory setae, with few setae forming posterior row along pigmented section and several setae medially; sternite 7 same but setose throughout; sternite 8 with small patch of pigmented area or undifferentiated from surrounding membrane. Ovipositor ( Figure 4E): Segment 8 with large lateral group of straight to slightly curved setae on prominent sockets. Segment 9 with pigmented rod-like sclerite along segment. Cercal segment in straight angle relative to segment 9, with ventral field of setulae, strongly sclerotized lateral plate harboring dozens of curved setae on prominent sockets to mid-length, group of shorter setae extending ventrally to unpigmented area, and eight to ten conspicuously long, curllike setae on distal half, followed by field of shorter, upright acicular setae extending to apical lamella; apical lamella short-cylindrical to rectangular, barely longer than lateral plate, setose and setulose.
Larva (third instar): Light yellow to bright orange. Integument covered by verrucae. Spatula bidentate, conspicuous, with well-developed shaft in some species (e.g., Figure 5C) to vestigial and barely visible in others. Sternal papillae usually asetose; four lateral papillae, asetose or two of which with very short setae, pleural papillae with distinct setae; three terminal papillae on each side, with long setae ( Figure 5D).
Pupa: Light to dark orange. Antennal bases well developed into tapered horns, parallel or splayed; no facial horns or papillae; short cephalic setae on elevated bases; prothoracic spiracle short and wide, with trachea forming loop inside spiracle ( Figure 3F). Basal part of facial area on each side with conspicuous lobe extending laterally. Dorsum of abdominal segments with wide transverse fields of tapered spicules across mid-section ( Figure 3G).
Etymology: The genus name is feminine, combining Ruschia Schwantes, a plant genus of 213 leaf succulent, perennial, shrubby species [1], which hosts the greatest diversity of gall midge species among the Aizoaceae, with the diminutive suffix 'ola'. Larva (third instar): Light yellow to bright orange. Integument covered by verrucae. Spatula bidentate, conspicuous, with well-developed shaft in some species (e.g., Figure  5C) to vestigial and barely visible in others. Sternal papillae usually asetose; four lateral papillae, asetose or two of which with very short setae, pleural papillae with distinct setae; three terminal papillae on each side, with long setae ( Figure 5D).
Pupa: Light to dark orange. Antennal bases well developed into tapered horns, parallel or splayed; no facial horns or papillae; short cephalic setae on elevated bases; prothoracic spiracle short and wide, with trachea forming loop inside spiracle ( Figure 3F). Basal Gall and biology: This species develops in swollen, succulent leaf galls, usually 2-6 cm long and 1-1.5 cm wide ( Figure 6A-D). The galls may be green but are usually reddish, occasionally with longitudinal cracks on most of the leaf area. Each gall may contain 20-30 larvae in vaguely defined, tunnel-like chambers along the longitudinal axis of the leaf, the walls of which are drier than the surrounding succulent tissues encompassing the rigid middle part of the gall. Galls on Lampranthus haworthii are often smaller and less conspicuous than those on Ruschia caroli and R. pungens, and the larval chambers in them are embedded in rigid, black tissue (probably fungal mycelia). The galls are heavily parasitized by several parasitoid Hymenoptera, including polyembryonic Platygastridae. The species is very common, and its galls can be found in large numbers at some sites, especially on R. caroli. Adults emerged mainly in spring (August-September), but galls containing mature larvae and pupae were also found in April, suggesting that the species completes at least two generations a year during the fall and winter months. Full-sized galls were also observed on R. caroli in summer (January), but they contained only first-instar larvae. This suggests that the larvae that hatch from eggs in spring (September-October) spend several months in the plant tissues before the galls become apparent and take several additional months to mature and pupate. The galls on all three host plants yielded a second, much smaller Lasioptera-like species, whose smaller and more delicate larvae and pupae were found alongside the robust larvae and pupae of R. succulenta. This second lasiopterine appears to be an inquiline and will be treated in a future publication, together with congeners we reared from several other succulent Aizoaceae. Adult description: Head, thorax and lateral parts of abdomen white; legs black; wing surface covered by dark hair-like setae, with thick black setae on C up to meeting point with R4+5. Abdominal tergites almost entirely black, except for thin transverse line of white scales along posterior margin. Abdominal pleura white, each with small black spot.
Head ( Figure 3B): Gap between eyes on vertex one to two facets wide. Antennal flagellomeres 11-15 in female (n = 30), 10-12 in male (n = 16), number often differs between Adult description: Head, thorax and lateral parts of abdomen white; legs black; wing surface covered by dark hair-like setae, with thick black setae on C up to meeting point with R 4+5 . Abdominal tergites almost entirely black, except for thin transverse line of white scales along posterior margin. Abdominal pleura white, each with small black spot.
Female abdomen ( Figure 3D): Tergites 1-7 with anterior pair of minute sensory setae and posterior row of long setae; tergite 8 hardly differentiated from surrounding membrane, with small, irregular patch of pigmented area, or completely unpigmented, pair of sensory setae anterior to pigmented area the only vestiture ( Figure 4E). Sternites without proximal sensory setae; sternites 2-6 with unpigmented patch medially, group of long setae medially and posterior row of long setae; sternite 7 evenly pigmented, with long setae on distal two thirds; sternite 8 completely undifferentiated from surrounding membrane. Ovipositor ( Figure 4E): Segment 9 with pigmented patches proximally and strongly pigmented rodlike sclerite along segment. Cercal segment with heavily sclerotized lateral plate, slightly tapered apically but not differentiated into clear aculeus; lateral plate bearing about 30 long, curved setae up to mid-length, with additional group of 10-20, shorter setae extending ventrally to unpigmented area, and eight to ten conspicuously long, thick, erect, curl-like setae on distal half, followed by group of shorter, upright acicular setae to tip of lateral plate, extending ventrally to apical lamella; area of lateral plate ventral to curl-like setae bare, sheathing almost half height of apical lamella before abrupt attenuation toward tip. Apical lamella rectangular, barely longer than lateral plate, strongly setose and setulose along distal half. Base of cercal segment with group of strong setae proximal to setulose hypoproct, and densely setulose ventral area extending to tip of apical lamella.
Male abdomen ( Figure 3E): Tergites 1-7 as in female; tergite 8 much smaller, without posterior row of setae. Sternites 2-7 as in female, pigmented area of sternite 8 smaller than preceding. Terminalia ( Figure 5A): Gonocoxite widest at mid-length, with numerous strong setae more or less evenly distributed; mediobasal lobe much shorter than aedeagus, almost same width throughout length or slightly narrows to apex in dorsal view, apex rugose, with multiple short setae on bulging bumps. Gonostylus widest at proximal third, setulose on proximal half and furrowed beyond both dorsally and ventrally, with wide apical tooth and numerous long, evenly distributed setae. Aedeagus wide, truncated apically in dorsal view, triangular and pointed anteriorly in lateral view. Hypoproct entire, truncated, or with very shallow apical depression, two setae apically and setulose throughout. Cerci robust, strongly setose and setulose.
Larva (third instar): Antennae about twice as long as wide. Posterolateral apodemes at least twice as long as head capsule ( Figure 5B). Spatula ( Figure 5C) well developed, with short, apically rounded teeth separated by round notch; long, narrow shaft, widens posteriorly. On each side of spatula asetose sternal papilla, four lateral papillae, two of which with minute setae, and one asetose ventral papilla; pleural and dorsal papillae with long setae. Terminal abdominal segment ( Figure 5D) on each side with three papillae bearing long setae; anus ventral.

Ruschiola attenuata Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plant: Mesembryanthemum splendens L. Gall and biology: This species induces common inflated, succulent leaf galls, typically up to 2 cm long and 0.5 cm wide ( Figure 6E,F). The gall usually occupies the entire leaf, is green, yellowish or reddish, and contains 1-3 chambers located centrally along the longitudinal leaf axis. The walls of the chambers are drier than the surrounding leaf tissue and the gall is firm to the touch. Galls are common throughout the distribution of the host plant and adult gall midges were reared from January to September, suggesting that the species completes multiple generations a year.
Female abdomen: Tergite 8 with small patch of pigmented area on each side, with anterior sensory seta and sometimes 2-3 small posterior setae. Ovipositor ( Figure 8E): Segment 9 with rectangular patch of pigmented area at mid-length. Heavily sclerotized area of lateral plate ventral to curl-like setae thin, sheathing at most quarter height of apical lamella. Empodia as long as or slightly longer than bend in claws ( Figure 8C).
Female abdomen: Tergite 8 with small patch of pigmented area on each side, with anterior sensory seta and sometimes 2-3 small posterior setae. Ovipositor ( Figure 8E): Segment 9 with rectangular patch of pigmented area at mid-length. Heavily sclerotized area of lateral plate ventral to curl-like setae thin, sheathing at most quarter height of apical lamella.
Larva: Not studied. Pupa (Figures 7B and 9A): Antennal horns straight, separated by triangular gap, each split apically into longer lateral tip and shorter median tip. Face with two tiny pits in mid posterior area. Pupa (Figures 7B and 9A): Antennal horns straight, separated by triangular gap, each split apically into longer lateral tip and shorter median tip. Face with two tiny pits in mid posterior area.  Distribution: Common wherever the host plant was found, outside or along the edge of winter rainfall areas of the Western Cape. Collected from Worcester, Laingsburg, Van Wyksdorp, and Oudtshoorn.
Etymology: The species epithet is the Latin feminine adjective term for plain, shortened or refined, with reference to the thinner and less robust lateral plate of the ovipositor compared to the ovipositor of other known Ruschiola species. Comments: This is the only Ruschiola species that can be readily distinguished from the otherwise rather uniform species in the genus based on its adult morphology. Its flagellomeres are more numerous and significantly shorter than those of the other species described here, the empodia are about as long as the bend in the claws rather than much longer, as in other species, and the ovipositor has a thinner lateral plate and less robust apical lamella. The pupa also stands out among all other species for having apically divided antennal horns, whereas those of all other species are undivided.
Gall and biology: This species induces conspicuous but uncommon sausage-like leaf galls on R. cymosa, R. cf. caroli and R. cf. cedarbergensis ( Figure 10A,B,D), whereas on the low, crawling R. schollii, it develops in reddish sections of otherwise undeformed leaves ( Figure 10C). The conspicuous galls are usually 5-7 cm long and at least 1 cm wide, often with longitudinal cracks, and may occupy the entire leaf (the common case) or only parts of it. They are firm to the touch and contain many larvae that develop in vaguely defined larval chambers embedded in rigid tissues. Galls were collected in August and September only; it is therefore unknown if this species has more than one generation per year.
Adult: Head: Antennal flagellomeres 12-14 in female (n = 24), 11-12 in male (n = 10); occasionally two or more flagellomeres fused to form continuous units. Palpus often appears composed of one large, globular segment, but usually with vestigial second segment "riding" on or branching from it ( Figure 11A). Insects 2022, 13, x FOR PEER REVIEW 21 of 36  Adult: Head: Antennal flagellomeres 12-14 in female (n = 24), 11-12 in male (n = 10); occasionally two or more flagellomeres fused to form continuous units. Palpus often appears composed of one large, globular segment, but usually with vestigial second segment "riding" on or branching from it ( Figure 11A). Male abdomen: Gonostylus ( Figure 8C) widest at base, with clear constriction around mid-length, distal margin almost straight rather than arched, setulose along basal and most of distal sections, furrowed elsewhere.
Larva (third instar): Antennae about twice as long as wide. Spatula ( Figure 11B) only slightly longer than wide, shallow teeth rounded apically, short and wide shaft extending into two lobes posteriorly. Lateral papillae asetose. One larva found with long-shafted spatula similar to that of R. succulenta; more sampling is needed in order to clarify if the short-shafted larvae represent early third instars with spatula that is not yet fully developed. Male abdomen: Gonostylus ( Figure 8C) widest at base, with clear constriction around mid-length, distal margin almost straight rather than arched, setulose along basal and most of distal sections, furrowed elsewhere.
Larva (third instar): Antennae about twice as long as wide. Spatula ( Figure 11B) only slightly longer than wide, shallow teeth rounded apically, short and wide shaft extending into two lobes posteriorly. Lateral papillae asetose. One larva found with long-shafted spatula similar to that of R. succulenta; more sampling is needed in order to clarify if the short-shafted larvae represent early third instars with spatula that is not yet fully developed.
Distribution: Uncommon on several Ruschia species restricted to the Cedarberg region. Recorded from Heuningvlei, Bushmans Kloof Wilderness Reserve, and Travellers Rest (Wolfdrif) near the town of Clanwilliam.
Etymology: The species epithet is feminine, combining the name Cedarberg with the Latin adjectival suffix "ensis", which means "from the Cedarberg", as it was only reared from several host plants in that particular region. Comments: The large, succulent leaf galls of this species on most of its host plants are very similar to those caused by R. succulenta and the two species appear to be closely related based on molecular data ( Figure 2). Nevertheless, slight diagnostic morphological differences do exist between them, including the shorter and more robust palpus in R. cedarbergensis, the typical shape of the gonostylus (Figure 11C), the much shorter and wider larval spatula and the more widely separated pupal antennal horns (parallel in R. succulenta). It is noteworthy that R. cedarbergensis causes large, conspicuous galls on three of its host plants, whereas on Ruschia schollii, no obvious galls develop, and the presence of the gall midge in the plant is evident only because of pupal skins that remain stuck in reddish sections of the otherwise undeformed leaves ( Figure 10C).

Ruschiola namaqua Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plants: Ruschia viridifolia L.Bolus, and R. goodiae L.Bolus. Gall and biology: This species develops in smooth, succulent, reddish leaf galls that usually occupy most of the leaf area ( Figure 10E,F). The galls are 3-4 cm long and 1.0-1.5 cm wide, each containing numerous larval chambers, and can be locally very common. They were sampled at several sites in late July to mid-August, at which time they contained second and third instar larvae, pupae, and empty pupal skins. It is currently unknown if this species has more than one generation a year.
Adult: Head: Antennal flagellomeres 12-13 in female (n = 39), 10-12 in male (n = 35). Palpus usually one segmented, either globular or with tapered extension, sometimes with basal bulge, occasionally two segmented, with tapered second segment much longer than first ( Figure 12A). basal bulge, occasionally two segmented, with tapered second segment much longer than first ( Figure 12A).  Etymology: This species is named after the Namaqualand region, to which it is apparently restricted. The name is a feminine noun in apposition. Comments: The adults and pupae of this species are virtually indistinguishable from those of R. succulenta, whereas the larvae have a greatly reduced spatula relative to the welldeveloped spatula of R. succulenta and setose rather than asetose sternal papillae. The galls are smaller, less rigid and without longitudinal cracks, which are typical of R. succulenta, R. cedarbergensis and other (undescribed) species from other host plants in Namaqualand.

Ruschiola bubonis Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plant: Jordaaniella spongiosa (L.Bolus) H.E.K. Hartmann. Gall and biology: This species develops without obvious gall formation in the massive, fleshy leaves of the host plant, and was discovered in the field only due to the empty pupal skins stuck in the leaves ( Figure 13A,B). Therefore, plant material was collected haphazardly in an attempt to rear the gall midges, a method that proved successful. Several individuals develop in the same leaf, and the species probably has more than one generation per year, at least during winter, as adults were reared in mid-July and mid-August. Adult: Head: Antennal flagellomeres 12-14 in female (n = 10), 11-13 in male (n = 5). Palpus morphology variable; occasionally one segmented but usually two segmented, with second segment either shorter or longer than first segment, with long scales ( Figure  14A). Adult: Head: Antennal flagellomeres 12-14 in female (n = 10), 11-13 in male (n = 5). Palpus morphology variable; occasionally one segmented but usually two segmented, with second segment either shorter or longer than first segment, with long scales ( Figure 14A). Male abdomen: Pigmented area of tergite 8 narrow, band-like or much shorter than preceding. Terminalia ( Figure 14B): Aedeagus narrow, parallel sided and apically truncated in dorsal view. Hypoproct entire.
Larva (third instar) ( Figure 14C): Posterolateral apodemes about as long as head capsule. Spatula virtually absent, evident only as small and vaguely defined pigmented area. On each side asetose sternal papilla and four asetose lateral papillae, one of which set closer to median line of body, the other three grouped together. Pleural, dorsal and terminal papillae with short setae. Male abdomen: Pigmented area of tergite 8 narrow, band-like or much shorter than preceding. Terminalia ( Figure 14B): Aedeagus narrow, parallel sided and apically truncated in dorsal view. Hypoproct entire.
Larva (third instar) ( Figure 14C): Posterolateral apodemes about as long as head capsule. Spatula virtually absent, evident only as small and vaguely defined pigmented area. On each side asetose sternal papilla and four asetose lateral papillae, one of which set closer to median line of body, the other three grouped together. Pleural, dorsal and terminal papillae with short setae.
Pupa (Figures 7E and 9D): Antennal horns slightly ventrally arched, widely separated at base, abruptly splayed along median margins from mid-length to tapered apex.
Distribution: Locally common on its host plant in the Namaqua National Park (coastal section), the only site where the plant was sampled.
Etymology: The epithet bubonis is the feminine genitive form of bubo-the Latin term for owl-with reference to the splayed antennal horns of the pupa, giving it the appearance of a horned owl ( Figure 7E). Other material examined: 1 larva, 3 exuviae, same data as holotype.
Comments: This species stands out among congeners with known pupae for the splayed, owl-like antennal horns of its pupa compared to the triangular or parallel-sided antennal horns of other species. Females cannot be distinguished from those of other species, with the exception of R. attenuata, but males have a consistently entire hypoproct, whereas the shape of the hypoproct in other species varies. The single available larva lacks a distinct spatula, and its pleural, dorsal and terminal papillae bear much shorter setae than those of other species.

Ruschiola quagga Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plant: Ruschia holensis L. Bolus. Gall and biology: This species develops in inflated leaf galls, 2-3 cm long and about 1 cm wide, green to reddish ( Figure 13C). Each gall contains a few larvae. In young galls containing second instar larvae, the larvae are found in vaguely differentiated chambers embedded in the spongy leaf tissue. Third instar larvae and pupae are found in more defined chambers with drier walls compared to the juicy surrounding tissues. The galls are heavily parasitized by polyembryonic hymenopteran parasitoids. Galls were collected only once, in late August, at which time they contained either second or third instar larvae and pupae. It is unknown if the species has more than one generation per year.
Adult: Head: Antennal flagellomeres 13 in female (n = 3), unknown in male (no males with complete antennae). Palpus one segmented, 1.3-5.5 times as long as wide, usually tapered ( Figure 14D); when short, tear shaped and based on small palpiger; shape may differ between palpi of same individual. Thorax

Ruschiola timida Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plant: Scopelogena bruynsii Klak. Gall and biology: This species is rare, or at least very difficult to find. It develops without causing any obvious deformation in the leaves of its host plant and was reared only by collecting plant material haphazardly after finding a small number of empty pupal skins stuck in leaves ( Figure 13D). On one occasion, a slightly inflated leaf was found, but all adults that we reared emerged from leaves with no external signs of infestation. Adults were reared only from one site, although we sampled the host plant at other sites within the same area, where the plant is rather common. In 2017 and 2018, plants were collected and adults emerged from them in mid-September; in 2019 plants were sampled in early August, but no adults emerged. Additional sampling of the host plant is needed at different times of the year to confirm if the species has only one generation in early spring.

Ruschiola furtiva Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plant: Ruschia dichroa (Rolfe) L. Bolus. Gall and biology: This species develops in inconspicuous leaf galls, the only external deformation of the leaf being a slight change of color from green to red. The presence of the gall midges in the leaves was recognized only by the empty pupal skins that were found stuck in slightly discolored leaves. Although the same site was visited twice in subsequent years, and host plant material was collected, gall midges were reared only once, in mid-September; no other information is currently available on the life history of this species.

Ruschiola leipoldtiae Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plant: Leipoldtia laxa L. Bolus and L. schultzei (Schltr. and Diels) Friedrich. Gall and biology: This species induces common leaf galls on its host plants, which occupy either the entire leaf or only part of it ( Figure 15A-D). The galls are pinkish-red, rigid, and contain multiple larval chambers (10-20 on L. schultzei, fewer on L. laxa) embedded in a mass of tough tissue in the center of the fleshy leaf. Galls were found and midges were reared in late July and early August. No additional information is available on the life history of this species; however, in December no galls were found on L. laxa at the same site from which they were collected in August of the previous year; hence, the species appears to be inactive during the summer.
Distribution: This species was found only once in Bushmans Kloof Wilderness Reserve in the Cedarberg region, to which it is probably endemic, similar to its host plant [40].
Etymology: The species epithet is a feminine Latin adjective for secret or hidden, referring to the lack of external signs of its presence in the leaves of its host plant. Other material examined: 8 exuviae, same data as holotype.

Ruschiola leipoldtiae Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plant: Leipoldtia laxa L. Bolus and L. schultzei (Schltr. and Diels) Friedrich. Gall and biology: This species induces common leaf galls on its host plants, which occupy either the entire leaf or only part of it ( Figure 15A-D). The galls are pinkish-red, rigid, and contain multiple larval chambers (10-20 on L. schultzei, fewer on L. laxa) embedded in a mass of tough tissue in the center of the fleshy leaf. Galls were found and midges were reared in late July and early August. No additional information is available on the life history of this species; however, in December no galls were found on L. laxa at the same site from which they were collected in August of the previous year; hence, the species appears to be inactive during the summer. Adult: Head: Antennal flagellomeres 11-13 in female (n = 26), 11-12 in male (n = 10). Palpus one or two segmented ( Figure 16A,B), number sometimes varies in same individual; when one segmented, segment ovoid, 1.5-2.0 times as long as wide; when two segmented, segment 2 subequal to or longer than segment 1 and appears to "ride" on it ( Figure 16B). Labella prominent and elongated, 1.5 times as long as wide ( Figure 16A). Adult: Head: Antennal flagellomeres 11-13 in female (n = 26), 11-12 in male (n Palpus one or two segmented ( Figure 16A,B), number sometimes varies in same ind ual; when one segmented, segment ovoid, 1.5-2.0 times as long as wide; when two mented, segment 2 subequal to or longer than segment 1 and appears to "ride" on it ure 16B). Labella prominent and elongated, 1.5 times as long as wide ( Figure 16A). Male abdomen: Gonostylus widest at proximal third, distal margin almost straight rather than arched ( Figure 16D). Hypoproct truncated or with shallow notch.
Larva (third instar): Posterolateral apodeme about as long as head capsule. Spatula well developed, long shafted ( Figure 16C). On each side of spatula asetose sternal papilla and 4 asetose lateral papillae, one of which set further apart from the other three.
Distribution: This species was found in Springbok and in the Namaqua National Park (Skilpad camp), suggesting that it has a wide distribution in Namaqualand.
Etymology: This species is named after its host plant genus Leipoldtia, which in turn is named after the renowned South African physician, poet and botanist, C. Louis Leipoldt (1880-1947). The epithet is a feminine noun in the genitive case. Comments: This species stands out among its congeners for its conspicuous labella and dense cover of hair-like setae on the adult frons and male gonopods. The gonostylus is straight along its posterior margin rather than arched, as in most species. The pupa resembles that of R. cedarbergensis in having antennal horns that are not parallel along their median margins (although this character depends on the angle from which the pupae are viewed and may be distorted in slide-mounted exuviae).

Ruschiola celebrata Dorchin and van Munster, New Species
Characters similar to R. succulenta, except for the following: Host plants: Mitrophyllum mitratum (Marloth) Schwantes, M. clivorum (N.E.Br.) Schwantes. Gall and biology: This species was reared from inflated leaf galls that usually occupy the base of the leaf, but which occasionally occupy its entire length ( Figure 15E,F). Often only one of the two leaves on the same node is affected ( Figure 15E). The galls were locally very common on the two host plants at a particular spot, but absent from other spots at the same site. As would be expected, galls were larger on M. mitratum (2-4 cm) than on the smaller and more delicate M. clivorum (1-2 cm). Each gall contained 10-30 larvae, as attested by the large numbers of empty pupal skins that were stuck in them. The galls are not particularly harder than the soft, uninfested leaves, and most are green but sometimes yellowish. This species was observed and reared only once, in late July, and no additional information is available on its life history.
Etymology: The species epithet is a feminine Latin adjective for crowded or populous, with reference to the large number of larvae in the gall. Comments: The pupa of this species is unique among other known pupae in this genus for having a small but distinct bulge just below the apex of the antennal horn (compare Figure 16F,G).

Discussion and Conclusions
In this study we revealed a species-rich community of gall midges on southern African Aizoaceae, which was unknown to date. While it was obvious that these gall midges belong to the Lasiopterini, they were morphologically distinct from all other known genera in that tribe, justifying the description of a new genus. Our findings attest to the vast gap in the knowledge on Afrotropical Cecidomyiidae and on the diversity of Lasiopterini in particular, the greatest diversity of which is currently known from Chenopodiaceae host plants in the Palaearctic region [9,21,41]. The phylogenetic relations of Ruschiola to other genera in the Lasiopterini require further study.
The general morphological uniformity among Ruschiola species may indicate a recent diversification on succulent Aizoaceae, independently from the parallel diversification of Asphondylia species on these host plants. This parallel diversification was probably aided by the fact that the two taxa occupy different niches on the host plants, with the Ruschiola species developing in leaves and the Asphondylia species in buds. Most of the Ruschiola species described here are highly host specific, having been recorded from one or a few closely related host-plant species, as seen in Lasiopterini from Chenopodiaceae (e.g., [38,42]). However, the two groups differ in that chenopod-associated taxa are much more diverse morphologically and that each chenopod host often supports multiple rather than a single lasiopterine species (e.g., [43][44][45]), including in South Africa (N. Dorchin, unpublished data). These observations suggest that the association of Lasiopterini with Chenopodiaceae is older than their association with Aizoaceae.
The phylogenetic inference presented here implies that the cecidomyiid species associated with Ruschia spp. are more closely related to each other than to species from other host plant taxa, although these results should be taken with caution given that they are based on a single mitochondrial gene. They also attest to a strong geographic effect, by which the same cecidomyiid species uses several host plant taxa that are found in physical proximity (e.g., R. cedarbergensis and R. namaqua on several Ruschia species in the Cedarberg and Namaqualand regions, respectively). Lastly, our findings provide yet another example of the importance of immature stages in studies of taxonomically difficult cecidomyiid groups, as larvae and pupae may provide diagnostic characters that are absent in adults (e.g., [46][47][48]).   Table 1. Other data are available upon request from the corresponding author.