﻿Calothecanigromaculata species-group from sub-Saharan Africa with descriptions of two new species from KwaZulu-Natal (Chrysomelidae, Galerucinae, Alticini)

﻿Abstract Calotheca Heyden is a flea beetle genus with a largely sub-Saharan distribution and currently comprising 34 species. The examination of new material is revealing an increase in species richness and intraspecific variability. Calothecacarolineaesp. nov. and C.wanatisp. nov., both from KwaZulu-Natal in the Republic of South Africa, are here described and attributed to the C.nigromaculata (Jacoby) species group, mainly based on genitalic characters. Photographs of the main diagnostic characters are provided, including the habitus, median lobe of the aedeagus, and spermatheca. Information on the geographic distribution and host plants of these species is also provided.


Introduction
Calotheca Heyden, 1887 is a flea beetle genus (Chrysomelidae, Galerucinae, Alticini) that is widespread in sub-Saharan Africa where is particularly common in the eastern and southern parts, with some records from Israel and the Arabian Peninsula D'Alessandro 2010a, 2012;Biondi et al. 2017;D'Alessandro et al. 2018a). Up to now this genus comprised 32 species, mainly associated with plants in the family Anacardiaceae, particularly Searsia spp., which are distributed in several different forest and savannah environments (D'Alessandro et al. 2018a(D'Alessandro et al. , 2020Iannella et al. 2021). The new material under examination is revealing an increase in species richness and intraspecific variability. Since the knowledge of the Afrotropical flea beetle fauna is far from exhaustive, unexpected diversity is often discovered within genera (e.g. D'Alessandro 2008, 2010b), new genera are recognized (e.g. Biondi and D'Alessandro 2013a), or genera are recorded for the first time from the Afrotropical region (e.g. Biondi and D'Alessandro 2013b). Calotheca was identified as a monophyletic group based on a phylogenetic analysis which included Blepharida rhois (Forster, 1771) from North America among the outgroups (Biondi et al. 2017). Even though the Afrotropical species of Calotheca are the most closely related to the North American B. rhois, they are significantly separated from it, and supported by two synapomorphies: frontal grooves sinuate and deeply impressed, extending approximately from the dorsal ocular margin to the interantennal space, and femora strongly punctured. The main diagnostic characters for Calotheca, compared to the closely related African genus Blepharidina Bechyné, 1968, are the sinuate and deeply impressed frontal grooves, extending approximately from the dorsal ocular margin to the inter-antennal space; and the punctate lateral striae on the pronotum, which run from the anterior margin onto the disc, and are straight, curved, or L-or C-shaped. Some species also have short lateral longitudinal furrows and/or small dimples close to the pronotal base (Biondi et al. 2017D'Alessandro et al. 2018bD'Alessandro et al. , 2019. In this paper we review, based on newly examined material, the distribution and ecological data of Calotheca nigromaculata (Jacoby, 1888) from southern Africa. We also describe two new species, Calotheca carolineae sp. nov. and C. wanati sp. nov., both from KwaZulu-Natal in the Republic of South Africa. The three species are attributed to the Calotheca nigromaculata species group, based on the morphology of the pronotum and spermatheca.

Materials and methods
Material examined consists of 212 dried pinned specimens, preserved in the institutions listed below in the "Abbreviations" section. The specimens were examined, measured, and dissected using a Leica M205C stereomicroscope. Photographs were taken using a Leica DFC500 camera and compiled using Zerene Stacker software, v. 1.04. Scanning electron micrographs were taken using a Hitachi TM-1000. Terminology follows D' Alessandro et al. (2016) for the median lobe of aedeagus and spermatheca.
Geographic coordinates for the localities were reported in degrees and minutes format using the WGS84 datum; information included in square brackets were added to the label data by the authors and using the Google Earth website for coordinates and geographic information. Abbreviations for the depositories follow the list on the following website: The Insect and Spider Collections of the World (Evenhuis 2021). Chorotypes follow Biondi and D'Alessandro (2006). proximal 1/2 and distinctly sinuate in apical 1/2 in lateral view, and produced and subtruncate apically in ventral view; in C. wanati sp. nov. it is curved in proximal 1/2 and more distinctly curved in apical 1/2 in lateral view, and produced and sub-rhomboidal in the apical 1/4, with prominent angulate lateral projections in ventral view). Other differences include: color of the dorsal integument with more clearly defined and generally larger colored elytral patches, reddish-brown on a yellow background (smaller and more confused, from brown to black, in C. nigromaculata and C. wanati sp. nov.) (Figs 1A, 2A-C, 3A); punctate lateral pronotal striae less dark (Figs 1A, 2A-C, 3A); antennae generally longer with LAN/(LE+LP) = 0.48 ± 0.01 in male, and = 0.41 ± 0.01 in female (in C. nigromaculata LAN/(LE+LP) = 0.42 ± 0.02 in male, and = 0.38 ± 0.02 in female; in C. wanati sp. nov. LAN/(LE+LP) = 0.42 ± 0.02 in male, and = 0.37 ± 0.02 in female) (Fig. 4A); shape of the pronotum, straight laterally in basal 2/3 and abruptly incurved in apical 1/3 (slightly curved in basal 2/3 and distinctly incurved in apical 1/3 in C. nigromaculata and C. wanati sp. nov.) (Figs 1C,2D,3C).
Etymology. The specific epithet is a noun in the genitive case after our friend Caroline S. Chaboo (University of Nebraska-Lincoln, Nebraska, USA), one of its collectors and appreciated expert of chrysomelid Coleoptera.
Ecological notes. Adults were collected in November, January, and February, between 0-50 m a.s.l., on Allophylus natalensis (Sapindaceae) and Ozoroa obovata (Anacardiaceae). Figures 2A-F  Taxonomic remarks. Calotheca nigromaculata displays much variation in the number, shape, and color of the elytral patches ( Fig. 2A-C), and in some biometric ratios (e.g., LE/LP) (Fig. 4). However, pronotal shape, sculpture, and color are consistent and useful for identification ( Fig. 2A-D): lateral margins barely or not visible in dorsal view, more incurved in the anterior third; punctate lateral striae and basal furrows distinctly impressed and generally darker than the rest of the pronotal surface; pronotal margins mostly darkened. Median lobe of the aedeagus (Fig. 2F) in ventral view: lateral margins sinuate, but prominently rounded in apical 1/4, subtruncate apically; ventral surface with a pair of rounded lateral U-shaped depressions with a wrinkled surface in the apical half; surface clearly punctate in the apical 1/4; in lateral view, aedeagus curved in the basal 1/2 and distinctly sinuate in the apical 1/2; dorsal ligula short but clearly visible in lateral view, formed by a subtriangular, apically truncate median lobe, and two lateral lobes. The apical part of the median lobe shows considerable variability: in ventral view it is more or less sinuate laterally and more or less prominently rounded in apical 1/4, and more or less sinuate in lateral view. Spermatheca (Fig. 2E) globosely fusiform basally, sub-conical and generally dorsally orientated at the ductus attachment; distal part distinctly curved, generally about as long as the basal part, with a distinct appendix; ductus basally inserted, thickset, short, uncoiled, roughly U-shaped.
Description of the holotype (♂). Body elliptical in dorsal view (Fig. 3A), rather convex in lateral view; total length of the body (LB) = 6.40 mm; maximum pronotal width in the middle (WP = 2.98 mm); maximum width of elytra in the basal third (WE = 3.80 mm). Head pale brown, slightly darker along the frontal grooves; antennae pale brown, with antennomeres 6-11 slightly darkened (Fig. 3A); surface microreticulate and densely micropunctate, with evident setiferous punctures on most of the vertex and part of the frons; frontal grooves sinuate, very deeply impressed, extending from approximately the dorsal ocular margin to the inter-antennal space (Fig. 3C); eyes elliptical, clearly elongate; antennae shorter than half the body length (LAN = 2.75 mm;  (Fig. 3A, C) slightly convex, pale brown, punctate lateral stria and part of the margins darkened; distinctly transverse in dorsal view (LP = 1.40 mm; WP/LP = 2.13), sub-rectangular, with sides more distinctly incurved in the anterior third; surface microreticulate and micropunctate, with minute punctation; anterolateral surface with very shallow depressions; punctate lateral striae distinctly impressed, C-shaped, and slightly curved on the disc; some additional sparse punctures along the pronotal margins; basal furrows short and moderately incised; anterior, basal, and lateral margins evenly and finely bordered; lateral margins barely visible in dorsal view; anterior angles distinctly prominent, indistinctly swollen; posterior angles obtuse. Scutellum brown, sub-triangular. Elytra (Fig. 3A) elongate (LE = 4.90 mm; WE/LE = 0.78; LE/LP = 3.50), moderately rounded and indistinctly sinuate laterally, jointly rounded apically; slightly paler than pronotum, with irregular brown patches and brown punctation; lateral margin narrow, barely visible in dorsal view; elytral punctation arranged in single regular rows formed by distinctly impressed punctures; interstriae flat on the elytral disc, with finely microreticulate and sparsely micropunctate surface; humeral calli indistinctly raised. Macropterous. Basal pro-and mesotarsomeres clearly enlarged (Fig. 3A). Underside brown; apical abdominal ventrite without preapical sculptures or impressions. Median lobe of the aedeagus (LAED = 2.28 mm; LE/LAED = 2.15) (Fig. 3B) distinctly sinuate laterally in ventral view; in ventral view it has a sub-rhomboidal apical 1/4 truncate apically, and the prominent lateral projections are angulate; ventral surface with a pair of elongate lateral U-shaped depressions with a wrinkled surface in the apical half; surface distinctly punctate in the apical 1/4; aedeagus curved in the basal 1/2 and distinctly sinuate in the apical 1/2; dorsal ligula short but clearly visible in lateral view, formed by a subtriangular apically truncate median lobe, and two narrower lateral lobes.
Etymology. The specific epithet is a noun in the genitive case after Dr Marek Wanat (University of Wrocław, Poland), one of its collectors and esteemed expert of Coleoptera Curculionoidea. Ecological notes. Adults were collected in November, December, and January, between 5-500 m a.s.l., on Allophylus natalensis (Sapindaceae) on one occasion and in swamp forest during a different collecting event.

Discussion
Calotheca carolineae sp. nov., C. nigromaculata and C. wanati sp. nov. differ from the other known Calotheca species in that they share a combination of morphological characters, which are listed below. The basal part of the spermatheca is sub-fusiform; the area where the ductus is attached is roughly conical; the distal part is distinctly curved, elongate and about 2/3 or sub-equal to the basal part in length, with a distinct appendix; the ductus is basally inserted, quite thickset, short, uncoiled, and roughly U-shaped (Figs 1D, 2E, 3D). Ventrally the aedeagus has a pair of elongate to sub-rounded U-shaped depressions with a wrinkled surface in the distal 1/2, and distinct punctation towards the apex (Figs 1B, 2F, 3B). The pronotum shows distinct, but not expanded, lateral margins which are not thicker than the basal margin, barely or not visible in dorsal view, and more distinctly incurved in the anterior third; the punctate lateral striae, and sometimes the basal furrows, are darker in color than the rest of the pronotal surface (Figs 1C, 2D, 3C). The head has evident setiferous punctures on most of the surface of the vertex and at least on part of the frons (Figs 1C, 2D, 3C). Clear similarity in the shape of the median lobe of the aedeagus of C. nigromaculata and C. wanati sp. nov. reveals a closer affinity between these species than with C. carolineae sp. nov. In these two species, the aedeagus shows (Figs 2F, 3B): in ventral view the apical 1/4 sub-rhomboidal, truncate, or slightly rounded apically, with a strongly punctate surface; two lateral U-shaped depressions with a wrinkled surface in the distal half; in dorsal view a short ligula, with a slender median lobe and lateral lobes; in lateral view the apical part clearly sinuate. The main characters discussed above are reported below in the form of a key to species to facilitate the identification of the specimens.
Based on the available data, C. nigromaculata displays a wider distribution, that includes those of C. carolineae sp. nov. and C. wanati sp. nov. The species are even syntopic in some areas (C. nigromaculata with C. wanati sp. nov. in Ubombo Mountain Nature Reserve, and C. wanati sp. nov. with C. carolineae sp. nov. in Sodwana Bay), and they are associated with the same plant genus Allophylus (Sapindaceae), with C. nigromaculata also collected on Searsia (Anacardiaceae). Sapindaceae represents the first record of a host plant family other than Anacardiaceae. The two families belong to the same order Sapindales (The Angiosperm Phylogeny Group 2016), indicating a possible phylogenetically constrained host-use for Calotheca.

Key to species of the Calotheca nigromaculata group
The three species are distinguishable mainly by the characters of the median lobe of the aedeagus. Females can be identified by evaluating the combination of: color pattern, which is consistent within Calotheca carolineae sp. nov. and C. wanati sp. nov. but variable in C. nigromaculata; pronotal shape; some biometric features, such as LAN/ LB and WE/LE.

Conclusion
The genus Calotheca currently comprises 34 species, including the two new species here described. Diagnostic characters at species level, based on morphology, are mainly found on the median lobe of the aedeagus, the pronotum, and in the color of the dorsal integument. The identification of particular species groups relies mainly on the characteristics of the spermatheca and pronotum (D'Alessandro et al. 2020Biondi unpublished data). Based on these characteristics C. carolineae sp. nov., C. nigromaculata, and C. wanati sp. nov. are here attributed to the C. nigromaculata species group. While the geographic distribution of the new species and the new distributional data of C. nigromaculata do not expand the geographic range of the genus, data on the association of the three species with the genus Allophylus (Sapindaceae) widen the range of its trophic spectrum, previously known as being limited to the family Anacardiaceae. However, due to the affinity between Anacardiaceae and Sapindaceae (The Angiosperm Phylogeny Group 2016), Calotheca species feeding on both the plant families cannot be considered as polyphagous.