Revision of the genus Coccidula Kugelann (Coleoptera, Coccinellidae)

Abstract The genus Coccidula Kugelann includes five species distributed in the Holarctic, with one species in North America and four in Palearctic region. Coccidula belongs to the tribe Coccidulini which historically was treated as a separate subfamily within ladybird beetles, but recent studies confirmed its placement as a tribe within the broadly defined subfamily Coccinellinae. All species are revised and a new synonymy of Lithophilus naviauxi Duverger with C. litophiloides Reitter is proposed. Light and electron microscopy pictures support morphological descriptions. An identification key to all species is also provided.


Introduction
The classification of ladybird beetles (Coccinellidae) has changed dynamically in the last decade mainly due to molecular approaches. Although several studies have been conducted at the family level, none of them gave robust classification of the family Robertson et al. 2015). Historically, the family was divided into six or seven subfamilies (Sasaji 1968;Kovář 1996) but more recent treatments based on morphology and molecules support just two, Microweiseinae and Coccinellinae (Ślipiński 2007;Seago et al. 2011;Robertson et al. 2015). However, recent analysis of a large molecular dataset (Che et al. 2021) revealed the existence of the third monotypic subfamily Monocoryninae.
The genus Coccidula Kugelann, 1798 was traditionally placed in the subfamily Coccidulinae (Sasaji 1968;Kovář 1996), nonetheless, in the new classification of ladybirds it was proposed to be one of the tribes (Coccidulini) within the broadly defined subfamily Coccinellinae. Seago et al. (2011) synonymized this tribe with Scymnini, however, after the analyses by Robertson et al. (2015) and Che et al. (2021) both are once again treated as independent tribes. Coccidulini are one of the most problematic groups of ladybirds as in the traditional classification they contain numerous genera with just superficial external similarity based mainly on hairy body surface and relatively long antennae. Consequently, in all molecular analyses they do not form a monophyletic group Robertson et al. 2015;Che et al. 2021). The tribe is distributed worldwide with moderate diversity in the Palearctic (Kovář 2007) and African regions (Fürsch 2007;Tomaszewska 2010), rich in South America (Gordon 1994), but the most diverse fauna occurs in South Asia, Australia and neighboring regions (e.g., Ślipiński 2007;Poorani and Ślipiński 2009;Tomaszewska 2010;Tomaszewska and Ślipiński 2011;Szawaryn and Leschen 2019). The largest and most widely distributed is the genus Rhyzobius Stephens, 1831 with more than 100 recognized species (Tomaszewska 2010;Czerwiński et al. 2020) and numerous undescribed species mainly from New Guinea. Interestingly it is also the only genus of Coccidulini with known fossil representatives from the Eocene period discovered in Oise (Kirejtshuk and Nel 2012) and Baltic ambers .
Coccidula is a small genus distributed in the Holarctic, with one species in North America and four in Eurasia. Historically numerous species and varieties have been described based mostly on differences in color pattern, but most of them were subsequently synonymized when genitalia were examined. Gordon (1985) revised the North American species; however, the Palearctic species have not been revised until now. As Coccidulini has never been a subject of morphological cladistic analysis there is no hypothesis about its internal relationship available. However, based on recent molecular analyses (Che et al. 2021), C. scutellata (Herbst, 1783) and Rhyzobius litura (Fabricius, 1787) group in a single clade with the African genus Epipleuria Fürsch, 2001 and African species of Rhyzobius.
The European species are usually found in wetlands and water banks in low and middle elevations (Bielawski 1959). They live on herbaceous emergent grassy vegetation such as reeds, feeding on aphids such as Hyalopterus pruni (Hemiptera: Aphididae). Coccidula rufa is sometimes contrastingly reported also from dry sand dunes and in Finland from cereal fields (Clayhills and Markkula 1974). High prevalence (60-80%) of endosymbiotic bacteria Rickettsia and Wolbachia was reported from Germany (Weinert et al. 2007). Coccidula rufa is univoltine -mating and egg-laying occur in the spring and summer, eggs are laid in batches on reed stems and foliage, larvae develop through the spring and summer, and a new generation of adults emerges in July. All species may be locally and temporally abundant.
The tribe Coccidulini needs comprehensive revision. In the current work we present a morphological revision of all currently known species of Coccidula, the type genus for the tribe. This revision is a first step to understand the morphological diversity of the tribe and may lead to further phylogenetic studies.

Material and methods
Material used of this study is deposited in the following collections: Genitalia were dissected, cleared in a 10% KOH solution, washed in water, and placed in glycerol on slides for further study. Female genitalia were stained with chlorazol black. Measurements were recorded as follows: TL -total body length from apical margin of clypeus to apex of elytra; PL -pronotal length from the middle of anterior margin to the middle of the posterior margin; PW -pronotal width across widest part; EL -elytral length along suture including scutellum; EW -elytral width across both elytra at the widest part. Colour images were taken using either a stereo microscope Leica MZ 16 with a digital camera IC 3D; final images were produced using Helicon Focus 5.0X64 and Adobe Photoshop CS6 software, or a stereo microscope Nikon SMZ 1500 with Lumenera digital camera and Quick-Photo software, composite images with deep focus were generated using Zerene Stacker. The SEM photographs were taken in the Laboratory of Scanning Microscopy, MIZ (Warsaw), using a scanning electron microscope HITACHI S-3400N under low vacuum conditions and on JEOL JSM-7401F in Biology Centre CAS (České Budějovice). Terminology used for morphology follows Ślipiński (2007) and Lawrence et al. (2011). In this paper, we follow the classification proposed by Che et al. (2021). Diagnosis. Representatives of the genus Coccidula with its general body shape may resemble Tetrabrachys Kapur, however, it can be separated based on the structure of the tarsi which are tetramerous in both genera but in Coccidula the first tarsomere is sub-triangularly broadened apically and the second is elongate and distinctly lobbed, while in Tetrabrachys both the first and second are narrow, elongate and without lobes. Moreover, in Tetrabrachys the apical maxillary palpomere is widely securiform, and beetles are brachypterous, while in Coccidula the apical maxillary palpomere is only slightly widened and the second pair of wings is functional. Coccidula is also externally similar to European species of Rhyzobius but it can be separated based on the following characters: body almost parallel sided, elytra covered with punctures of two sizes, larger punctures arranged in nine rows (in C. litophiloides some of them are reduced), base of the pronotum not bordered, while in Rhyzobius the lateral body outline is broadly rounded, the elytra are covered with single sized, randomly arranged punctures, and base of the pronotum with distinct bordering line.
Anterior margin of pronotum weakly, broadly emarginate (Figs 3B, 5B) with anterior corners broadly rounded; lateral margins with moderately (Figs 7B, 9B) to distinctly expanded lateral beads ( Body entirely rufous, sometimes with darker scutellar shield (Fig. 1G); penis guide distinctly curved in lateral view, parameres at base about as broad as in middle (Fig. 8B)  Diagnosis. Coccidula lepida is the only Nearctic species of the genus, and is similar in many characters to C. scutellata, but with the head and epipleura black. In the typical form (C. lepida described by LeConte), the black elytral pattern resembles an extension of the five fused black maculae on the elytra of C. scutellata, with shoulders and epipleura black. Shoulder tubercle distinct, prototum relatively narrow. Male genitalia with penis guide much shorter than parameres. Detailed description of morphology including variability in pattern can be found in Gordon (1985: 656-659).
Body elongate (Fig. 1C), slightly widening in posterior part. Head black. Elytra of typical form, light testaceous with black pattern covering scutellar shield and surrounding portion of elytra through shoulders to lateral margins, covering about 60% of its anterior part; pair of maculae in posterior 3/4 of elytra near suture; in western population fused and connected to scutellar shield over suture. Ventral side (Fig. 1D) black with hypomera and ventrites 3-6 testaceous.
Head and pronotum covered with uniform small setiferous punctures arranged irregularly. Pronotum transverse, broadly rounded laterally, with lateral margin glabrous; pronotum covered with dense setiferous punctures. Posterior pronotal corners not produced ( Fig. 2A). Prosternum with anterior margin with bordering line complete. Prosternal process with complete lateral carinae, joined roundly and merged with anterior border of pronotum (Fig. 2B).
Scutellar shield pentagonal, covered with dense setiferous punctures. Elytra (Fig. 2D) covered with two types of punctures, small setiferous punctures irregularly distributed throughout the elytral surface, some of these punctures surrounded by larger depressed circles forming nine irregular longitudinal rows along the whole length of elytra. Shoulder tubercles distinct, but lateral elytral margin of elytra visible from above throughout. Mesoventrite (Fig. 2C) with anterior border interrupted in median part. Metaventrite (Fig. 2C) with postcoxal lines transverse in median part and then descending laterally, continuous on the metaventral process in median part; covered with setiferous punctures very sparsely distributed in central part of sclerite, densely setose in anterolateral parts, with a single row of large punctures below postcoxal lines and above metacoxae.
Abdominal postcoxal lines (Fig. 2E) complete, widely rounded, reaching about half of the length of the ventrite 1 measured below metacoxa. Ventrites covered with dense setiferous punctures.
Male genitalia. Tegmen in inner view with penis guide subtriangular with pointed apex; short, about two times shorter than parameres. Parameres elongate elliptical, inner surface smooth, with long setae on the inner side and in apical margin. Penis simple with pointed apex. [see Gordon 1985: 657, fig. 539 a-d] Female genitalia. Sperm duct long, much longer than length of spermatheca. Spermatheca vermiform, broadest in basal part. [see Gordon 1985: 657, fig. 539e] Type locality. Vermont (USA). Distribution. North part of North America.  Diagnosis. Coccidula litophiloides is very distinctive among Coccidula species with large produced posterior pronotal angles, and a prosternal process without carinae (which are present in all remaining species). With its general body shape slightly widening posteriorly and pronotum distinctly widened laterally with broad lateral bead appearing glabrous, it is similar to C. scutellata. Male genitalia are distinctive with large, elliptical parameres possessing projections on their inner surfaces, which is also unique among Coccidula. Spermatheca, in female genitalia, is distinctly widening apically and has a very short sperm duct, about ¼ of the length of spermatheca.
Scutellar shield pentagonal, covered with dense setiferous punctures. Elytra covered with two types of punctures, small setiferous punctures irregularly distributed throughout elytral surface, some of these punctures surrounded by larger depressed circles, forming irregular longitudinal rows; rows 2 and 3 reduced or missing (Fig. 3A). Elytra more flattened in lateral view than in other Coccidula, without shoulder tubercle, lateral elytral margin visible throughout (Fig. 3A). Hind wings missing. Mesoventrite with anterior border complete. Metaventrite with postcoxal lines transverse, descending only laterally, fused on metaventral process in median part, forming continuous arc (Fig. 3D); covered with setiferous punctures very sparsely distributed in central part of sclerite, densely setose in lateral parts, without distinct rows of large punctures below postcoxal lines, large punctures above metacoxae present.
Abdominal postcoxal lines complete, rounded, reaching slightly less than half of length of the ventrite 1 measured below metacoxa. Ventrites covered with dense setiferous punctures.
Male genitalia. Tegmen in inner view with penis guide pentagonal with pointed apex (Fig. 4B); short, slightly longer than half length of parameres (Fig. 4A). Parameres large, elliptical, inner surface with distinct projections (Fig. 4B), with fringe of long setae in apical margin. Penis simple with pointed apex (Fig. 4C).
Type locality. Caucasus, Ordubad (Azerbaijan). Distribution. Armenia, Azerbaijan, Iran Remarks. Duverger (1983) described Lithophilus naviauxi from Iran. After examination of the type specimens (Fig. 1A, B) we noticed that this species does not belong to the genus Lithophilus Frölich (=Tetrabrachys Kapur). As drawn in the original publication (Duverger 1983), it has antennae with 11 antennomeres (10 in Tetrabrachys), and pseudotrimerous tarsi with tarsomere 3 very small and tarsomere 2 distinctly lobed, while in Tetrabrachys tarsi are distinctly tetramerous, with tarsomere 3 and 2 elongate, without distinct lobe. Duverger in his paper (1983) described L. naviauxi based on just three female specimens of which he illustrated the spermatheca (Duverger 1983: 89, figs 30, 31). However, C. litophiloides is also found in Iran. Comparison of the female genitalia of the type material of both taxa, and other available material, together with the lack of a second and third row of large punctures on the elytra, and other morphological features described in the original description of Duverger, led to the conclusion that L. naviauxi Duverger falls well within the definition of C. litophiloides; thus, we propose to synonymize both species.   Dodge, det. Merkl 1984/ prep. genital R. Bielawski 1956 (1 male: HNHM); Listvjanka pr. Bajkal, step, 29.6.1977, H. Karnecka lgt. (1 male, 1 female: NMP).
Diagnosis. Coccidula reitteri is very similar to C. rufa in external appearance, however, it can be distinguished by the presence of a small black transverse macula on the pronotum just anterior to the scutellar shield, and a longitudinal brown to black macula on the posterior half of the elytra on the elytral suture. Male genitalia are very close to C. rufa, however, the upper margin of the penis guide in lateral view is relatively less emarginated and parameres are narrower than in C. rufa.
Body elongate, parallel sided (Fig. 5A). Pronotum (Fig. 1F) with black transverse macula in front of the scutellar shield. Scutellar shield black. Elytra brown with elongate, dark brown to black macula along the elytral suture in posterior half. Ventral side testaceous with prosternum, mesoventrite, metaventrite, most of the ventrite 1 (except lateral corners), and central part of ventrite 2 black.
Head and pronotum covered with uniform small setiferous punctures arranged irregularly. Pronotum (Fig. 5B) transverse, broadly rounded laterally, with moderately broad, lateral margin without glabrous area; pronotum covered with dense setriferous punctures, with single row of larger punctures along lateral border. Posterior pronotal corners not produced (Fig. 5B). Prosternum with anterior margin with bordering line incomplete in median part, without small sub-rounded impression in center. Prosternal process with lateral carinae straight, joined together roundly at level of anterior border of procoxae, forming sub-triangular pattern (Fig. 5C).
Scutellar shield pentagonal, covered with dense setiferous punctures. Elytra covered with two types of punctures, small setiferous punctures irregularly distributed throughout the elytral surface, some of these punctures surrounded by larger depressed circles forming nine irregular longitudinal rows along whole length of elytra. Lateral elytral margin well visible throughout (Fig. 5A). Mesoventrite with complete anterior border. Metaventrite with postcoxal lines descending laterally, fused on metaventral process in median part, forming continuous arc (Fig. 5D); covered with setiferous punctures very sparsely distributed in central part of sclerite, densely setose in lateral parts, without distinct rows of large punctures below postcoxal lines, large punctures above metacoxae present.
Male genitalia. Tegmen in inner view (Fig. 6B) with penis guide broadly rounded in the median or apical part, with rounded apex; in lateral view (Fig. 6C) moderately expanded medially, with upper surface moderately emarginate; long, much longer than parameres. Parameres elongate, parallel sided, with narrow base, inner surface smooth, with fringe of long setae in apical part. Penis simple with pointed apex, with small bump before apex.

Distribution. Russia (East Siberia).
Remarks. Coccidula reitteri is very similar to C. rufa in external morphological characters as well as the structure of male and female genitalia (Fig. 6A) (Bielawski 1984); thus, further investigation, preferably of molecular markers, should be conducted to confirm whether it is a separate species or an eastern population of C. rufa. Figs 1A Reitter, 1900: 220. Coccidula rufa var. plagiata Gerhardt, 1910: 556. Material examined. Czech Rep., Zlín, 11.6.1999, lgt. L. Bureš (1: NMP);Mladá Boleslav, 25.4.1987, lgt. Nedvěd (1 male USB); Dvořiště, 9.8.1989, lgt. Nedvěd (1: USB); Kokořínský důl, 9.8.1995, lgt Diagnosis. Coccidula rufa is most similar in external appearance to C. reitteri, however it can be separated by the uniform testaceous coloration of the dorsal surface (C. reitteri possesses dark macula near the elytral suture). From uniformly colored specimens of C. scutellata it can be separated by the shape of carinae on the prosternal process. Male genitalia are also very distinctive: in C. scutellata penis guide is small, about half length of parameres, while in C. rufa it is longer than parameres. Spermatheca in female genitalia of C. rufa is vermiform, not widening apically, while in C. scutellata it is distinctly widened in apical part.
Head and pronotum covered with uniform small setiferous punctures arranged irregularly. Pronotum transverse, broadly rounded laterally, with moderately broad, lateral margin without glabrous area (Fig. 7B); pronotum covered with dense setiferous punctures, with a single row of larger punctures along lateral border. Posterior pronotal corners not produced (Fig. 7B). Prosternum with anterior margin with incomplete bordering line in median part, with a small sub-rounded impression in center. Prosternal process with lateral carinae straight, joined together roundly at level of anterior border of procoxae, forming sub-triangular pattern (Fig. 7C).
Scutellar shield pentagonal, covered with dense setiferous punctures. Elytra covered with two types of punctures, small setiferous punctures irregularly distributed throughout elytral surface, some of these punctures surrounded by larger depressed circles forming nine irregular longitudinal rows along whole length of elytra. Lateral elytral margin well visible throughout (Fig. 7A). Mesoventrite with complete anterior border. Metaventrite with postcoxal lines descending laterally, fused on metaventral process in median part, forming continuous arc (Fig. 7E), covered with setiferous punctures very sparsely distributed in central part of sclerite, densely setose in lateral parts, without distinct rows of large punctures below postcoxal lines, large punctures above metacoxae present.
Male genitalia. Tegmen in inner view ( Fig. 8A) with penis guide sub-parallel to broadly rounded, with rounded apex; in lateral view (Fig. 8B) expanded medially, with deeply emarginated upper margin; long, much longer than parameres. Parameres elongate, parallel sided, with just slightly narrower base, inner surface smooth, with fringe of long setae in apical part. Penis simple with sharply pointed and curved apex, with small bump before apex (Fig. 8C).
Female genitalia. Sperm duct long, longer than spermatheca (Fig. 8E). Spermatheca vermiform, not distinctly broadened apically. Accessory gland membranous, much shorter than sperm duct.  Coccidula scutellata var. aethiops Krauss, 1902: 92. Material examined. Armenia, Erevan, 9.06.1987, leg Diagnosis. Coccidula scutellata is the most variable species in body coloration. Typical forms with five black maculae on the elytra can be easily distinguished from other Coccidula species, however uniformly colored testaceous forms are externally similar to C. rufa. They can be easily distinguished by the shape of carinae on prosternal process, which are straight and form a sub-triangular pattern in C. rufa, and are sinuate and broadly rounded apically, and fused with anterior border of prosternum in C. scutellata. Moreover, C. scutellata has a more distinct shoulder tubercle, and relatively narrower protnotum. Also, the male genitalia are distinctive, with penis guide longer than parameres in C. rufa and much shorter in C. scutellata. Spermatheca, in female genitalia, is broadened apically in C. scutellata, while in C. rufa it is almost uniform in diameter.
Body elongate, slightly widening in posterior part. Elytra of typical (European) form testaceous with five black maculae (Fig. 1H), one large covering scutellar shield and surrounding portion of elytra, and four sub-oval maculae in the median part, two of which are placed close to elytral suture and remaining two, close to lateral margin. Sometimes macula surrounding scutellar shield extends along elytral suture, sometimes maculae placed in median part of elytra are fused, forming single band. Various forms with reductions of this pattern are also present to completely testaceous forms without any trace of black color. Ventral side testaceous with prosternal process, mesoventrite, metaventrite, most of ventrite 1 (except lateral corners), and central part of ventrite 2 black.
Head and pronotum covered with uniform small setiferous punctures arranged irregularly. Pronotum transverse, broadly rounded laterally, with broad, glabrous lateral margin (Fig. 9B); pronotum covered with dense setiferous punctures, with single row of larger punctures along lateral border. Posterior pronotal corners not produced. Prosternum with anterior margin with bordering line complete. Prosternal process with complete lateral carinae in form of sinuate line, joined roundly and merged with anterior border of pronotum (Fig. 9E).
Scutellar shield pentagonal, covered with dense setiferous punctures. Elytra covered with two types of punctures, small setiferous punctures irregularly distributed throughout elytral surface, some of these punctures surrounded by larger depressed circles forming nine irregular longitudinal rows along whole length of elytra. Shoulder tubercles distinct, lateral elytral margin of elytra not visible from above in anterior part (Fig. 9A). Mesoventrite with anterior border interrupted in median part. Metaventrite with postcoxal lines transverse in median part and then descending laterally, not fused on metaventral process in median part (Fig. 9D). Covered with setiferous punctures very sparsely distributed in central part of sclerite, densely setose in lateral parts, with single row of large punctures below postcoxal lines and above metacoxae. Abdominal postcoxal lines complete, rounded, reaching slightly more than half of length of ventrite 1 measured below metacoxa. Ventrites covered with dense setiferous punctures.
Male genitalia. Tegmen in inner view ( Fig. 10A) with penis guide subtriangular with pointed apex; short, about two times shorter than parameres. Parameres elongate elliptical (Fig. 10B), inner surface smooth, with long setae on inner surface and in apical margin. Penis simple with pointed apex (Fig. 10C).
Female genitalia. Sperm duct short, about as long as half of length of spermatheca (Fig. 10E). Spermatheca vermiform, distinctly broadened apically. Accessory gland membranous, longer than sperm duct.
Groszkowska (MIZ) is acknowledged for her help with the SEM illustrations, and Biology Centre CAS -The Laboratory of Electron Microscopy for making part of SEM images. Danny Haelewaters (USA), David Larson (USA), Vincent Nicolas (France), Gilles San Martin (Belgium) and Udo Schmidt (Germany) are acknowledged for providing habitus photos. The study was partially supported by grant number 20-10003S provided by the Grant agency of Czech Republic to O. Nedvěd. The reviewer and the Editor are acknowledged for their valuable comments on the earlier version of this manuscript.