A New Tribe of the Ladybird Beetle Subfamily Microweiseinae (Coleoptera: Coccinellidae) Discovered on an Island in the North Atlantic Ocean

Microweiseinae is a quite recently established subfamily within ladybird beetles (Coccinellidae). According to recent analyses of morphological and molecular data, it has been divided into three tribes. Members of the subfamily are distributed mostly in tropical and subtropical regions of the world. Despite several recent taxonomical studies of this group, its diversity and distribution is still not fully understood. Recent field collecting on Madeira Island resulted in the discovery of interesting specimens belonging to a yet unknown taxon, described here as Madeirodula atlantica gen. et sp. nov. Phylogenetic analysis of morphological characters indicate that the new taxon form a distinct branch within the subfamily Microweiseinae, for which we propose a new tribe Madeirodulini trib. nov. Evolutionary trends within the subfamily are discussed, and an updated key to the tribes of Microweiseinae is provided.


Introduction
Recent investigations of the Coccinellidae phylogeny significantly changed the classification of this group of beetles. Based on morphological data,Ślipiński [1] proposed dividing Coccinellidae into just two subfamilies, Microweiseinae and Coccinellinae, instead of six or seven previously proposed [2][3][4]. This point of view was later confirmed by subsequent molecular studies (e.g., [5,6]).
Members of the subfamily Microweiseinae have cryptic coloration and a very small body size, unlike most of the commonly known ladybird beetles with contrasting white, red, and black aposematic coloration. Their small, brown colored, pubescent body forms, more closely resemble some Anamorphidae, Eupsilobiidae, or Corylophidae than 'true' lady beetles. Recent molecular study of the superfamily Cucujoidea [6] places Microweiseinae as an intermediate clade between the remaining Coccinellidae and Endomychidae sensu lato (handsome fungus beetles).
Microweiseinae are regarded as a more primitive group of Coccinellidae, inhabiting mainly leaf litter or under bark habitats. Members of Microweiseinae inhabit mostly tropical and subtropical zones of the world, however, their distribution and life histories are largely unknown, and still many new species are described from different parts of the world [7][8][9][10][11]. The oldest representatives of this group are known from Eocene Baltic amber and belong to the tribe Serangiini [12,13].
Modern classification of Microweiseinae based on phylogenetic analysis of morphological characters [14] recognizes three tribes: Carinodulini Gordon et al., Serangiini Pope, and Microweiseini Leng, containing together about 150 species classified until the present study in 22 genera. The monophyly of the subfamily is well supported by several morphological characters of which the placement of antennal insertions well before the anterior margin of eyes, and asymmetrical tegmen of the male genitalia are unique modifications within the whole family Coccinellidae. Each tribe of Microweiseinae is quite distinct morphologically. The most apparent characters defining the tribes are: for Carinodulini, sublateral carinae on pronotum often associated with pits, and the V-shaped metaventral postcoxal lines; for Serangiini, mandible with a long dorsal process, antennal club large one-segmented and flattened, the prosternum raised and forming a triangular plate, and the ventral side of the body with distinct impressions for reception of legs; and for Microweiseini (the most diverse group of the subfamily), glandular structures on the subgenal area well delimited and separated from the mouth cavity and abdominal postcoxal lines descending and incomplete (although numerous exceptions/reversals of characters states are present in this tribe) [14].
The present study was inspired by a discovery of unusual new genus of the subfamily Microweiseinae, which was found on Madeira Island. It is described here as Madeirodula gen. nov., along with M. atlantica sp. nov. This is the first member of the Microweiseinae native to Europe (in terms of administrative boundaries), and it possesses a mixture of morphological characters present in all known tribes, especially of Carinodulini and Microweiseini. To test the systematic position of the new genus within the subfamily, a phylogenetic analysis was performed.

Type Specimen Deposition and Measurements
Specimens examined during this study were deposited in the following collections: NMPC, Natural History Museum Prague, Czechia, and JVC, the private collection of Jaroslav Větrovec, Hradec Králové, Czechia.
Measurements of the body structures were recorded as follows [15]: TL-Total body length, from the apical margin of clypeus to the apex of elytra; PL-Pronotal length, from the middle of the anterior margin to the margin of basal foramen; PW-Pronotal width in the widest part; EL-Elytral length along the suture, including scutellar shield; EW-Elytral width across both elytra in the widest part. Male genitalia were dissected, cleared in 10% KOH solution, and subsequently transferred in glycerol on slide for further study. After examination, the genitalia were glued on cards and pinned beneath the specimen. Digital photographs were made using a Leica MZ 16 stereo microscope with a digital camera IC 3D attached. Terminology used in this paper followsŚlipiński [1] and Lawrence et al. [16].

Taxon Sampling and Morphology Coding
Taxon sampling and characters used for the phylogenetic analysis based on Escalona and Slipiński [14], with terminals being the results of that study (considering the resulted synonyms). Additionally, two recently described genera of Microweiseinae were added to the dataset: Pangia Wang & Ren [8] and Ruthmuelleria Jałoszyński &Ślipiński [10]. The list of characters from Escalona andŚlipiński [14] was expanded with three additional characters. As a result, in our data matrix (Supplementary File S1), 22 currently recognized genera of the subfamily Microweiseinae and a new genus described here were scored for 48 multistate characters using DELTA (DEscription Language for TAxonomy) [17]. Two genera of Coccinellinae (Rhyzobius Stephens, Sticholotis Crotch) and one member of the family Corylophidae (Holopsis Broun) were coded as outgroups.

Phylogenetic Analyses
The matrix was exported to the Nexus format, checked in Mesquite [18] and exported to the TNT format for phylogenetic analyses. Unknown character states were coded with '?'. The maximum parsimony (MP) analyses were conducted in TNT 1.5 [19] using the Traditional Search option to find the most parsimonious trees (MPTs) under the following parameters: memory set to hold 1,000,000 trees, tree bisection-reconnection (TBR) branch-swapping algorithm with 1000 replications saving 10 trees per replicate; zero-length branches collapsed after the search, with implied weighting option with a k value set to 3. Bremer support was calculated using the TNT Bremer function, using suboptimal trees up to 20 steps longer. Character mapping was done in Winclada v1.00.08 [20] using unambiguous optimization. All characters were treated as unordered and analyses were performed under equal weights. The analysis was set to find the minimum tree length.
Additionally, the Bayesian inference (BI) was conducted in MrBayes v3.2.6 [21] running on the CIPRES Science Gateway v3.3. (phylo.org), using the Mkv model for standard data. All analyses used four chains (one cold and three heated) and two runs of 10 million generations. Autapomorphies were included in the dataset, and the analyses were conducted using a gamma distribution. Convergence of the two runs was visualized in Tracer v1.6 [22], and by examining potential scale reduction factor (PSRF) values and the average standard deviation of split frequencies in the MrBayes output.

Phylogenetic Analyses
The MP analysis under Traditional Search (MP TS) resulted in a single most parsimonious tree with topology parameters (L = 142; CI = 42; RI = 70) ( Figure 1A). The topology of the tree is similar to that presented as the preferred tree in Escalona andŚlipiński [14], with all known tribes of Microweiseinae: Carinodulini, Serangiini, Microweiseini, recovered. The differences refer to internal relationships between genera within the tribe Microweiseini, however, with Microfreudea Fürsch + Paracoelopterus Normand recovered as sister groups, and in the same position on the tree (as sister to the rest of Microweiseini) in both studies. In our BI analysis ( Figure 1B) Carinodulini are also recovered as a distinct clade, but Serangiini are embedded in Microweiseini and both tribes form a single clade with unresolved internal relationships. Interestingly, this placement of Serangiini in our BI analysis agrees with some variants of parsimony analysis from Escalona andŚlipiński [14]. In both present analyses (MP, BI), the new genus described here as Madeirodula gen. nov. was recovered as a sister taxon to the tribe Carinodulini, with its own apomorphies, enabling us to propose a new tribe of Microweiseinae.
be separated from Carinodulini by the prosternum forming a large chinpiece, abdominal postcoxal lines descending and incomplete laterally (vs. U-or V-shaped in Carinodulini), well developed hind wings, and by a lack of lateral carinae or pits on pronotum. From Serangiini, Madeirodulini can be distinguished by having an antennal club consisting of three antennomeres (vs. one antennomere), the mandible with broadly rounded, simple external border (vs. projected into a process in Serangiini), and the elytral epipleura without foveae for reception of legs.  Type genus. Madeirodula gen. nov., by monotypy and present designation. ZooBank. http://zoobank.org/urn:lsid:zoobank.org:act: Etymology. The tribal name is derived from the name of a type genus. Diagnosis. The new tribe Madeirodulini resembles members of the tribe Microweiseini in general body shape, but it can be separated from them by having a bidentate mandibular apex (vs. unidentate), antennae consisting of 11 antennomeres (vs. 7-10), cultriform apical maxillary palpomere (vs. conical, parallel sided or rounded), paired apophyses of male sternum IX joined apically in form of inverted V (vs. inverted Y), and by a lack of line separating anterior corners of pronotum from disc. Bidentate mandibles, antennae with 11 antennomeres, and cultriform apical maxillary palpomere are shared with members of the tribe Carinodulini, however, Madeirodulini can be separated from Carinodulini by the prosternum forming a large chinpiece, abdominal postcoxal lines descending and incomplete laterally Insects 2020, 11, 367 7 of 14 (vs. U-or V-shaped in Carinodulini), well developed hind wings, and by a lack of lateral carinae or pits on pronotum. From Serangiini, Madeirodulini can be distinguished by having an antennal club consisting of three antennomeres (vs. one antennomere), the mandible with broadly rounded, simple external border (vs. projected into a process in Serangiini), and the elytral epipleura without foveae for reception of legs.
Distribution. Europe, Macaronesia, Madeira ( Figure 5C).  Genus: Madeirodula gen. nov. (Figures 2-4). Type species. Madeirodula atlantica sp. nov., by monotypy and present designation. ZooBank. http://zoobank.org/urn:lsid:zoobank.org:act: Etymology. First part of the genus name is derived from the name of the island where the type specimens were collected, and the second part refers to Carinodula, the type genus of Carinodulini, sister group of the new tribe.
Diagnosis. Same as for the tribe. Description. Body elongate oval, flattened; dorsal surface pubescent ( Figure 2E,F). Madeirodula atlantica sp. nov. (Figures 2-4 Figure 2E,F), covered with double size setae, well visible long sparse setae, and very short and delicate setae that can be observed only under high magnification ( Figure 4C). Color chestnut brown, legs and mouthparts more pale. Eyes large, prominent, extending well beyond head capsule.
Prothorax. Pronotum transverse, without pits, with lateral sides narrowly and base comparatively widely margined; anterior margin with lines/bordering only in anterior angles, and median part not margined ( Figure 4B). Pronotal disc convex, covered with punctures of double size, larger punctures bearing large seta, sometimes with additional small puncture at its base; smaller punctures bearing small seta with second small puncture at its base; lateral edge smooth, sublateral carina absent ( Figure 4B). Prosternum with large, expanded laterally chinpiece ( Figure 3D); prosternal process parallel-sided, with rounded apex; prosternal carinae present, continuing along prosternal chinpiece and forming anterior prosternal border. Antero-median part of hypomeron with area of glandular structures or sensilla ( Figure 3E). Procoxal cavities transverse, with lateral slits ( Figure 3A).
Pterothorax. Elytra irregularly punctate with punctures of double size ( Figure 4A,C); larger punctures bearing large seta with additional large round impression at their base; smaller punctures bearing small seta with second small puncture at base; sutural line present only in apical part ( Figure 4A). Epipleura incomplete, narrow, without foveae, with short border line just in the mid length ( Figure 3A). Metathoracic wings well developed. Scutellar shield triangular, about as long as its width ( Figure 4C). Mesoventrite transverse ( Figure 4D), flat; with row of pores present on anterior raised margin; procoxal rest present; meso-metaventral junction arcuate anteriorly; at midline slightly broader than mid coxa. Metaventrite transverse ( Figure 4D), longer than ventrite 1; metaventral postcoxal lines joined medially at metaventral process forming straight line, laterally complete, descending; Metaventrite with rows of pores present under postcoxal lines and above hind coxae. Surface of metaventrite covered with small, sparsely distributed paired punctures (rarely single).
Legs slender (Figures 3A and 4F); coxae sub-rectangular with rounded inner angle, with a group of small pore openings at basal part; femora slightly swollen; tibiae without apical spurs; tarsi with three tarsomeres ( Figure 4E); tarsal claws with large basal, rectangular tooth.
Male genitalia. Tegmen asymmetrical with oblique cavity for reception of penis ( Figure 2G,H), parameres fused medially with rows of long setae at outer margin, penis guide with apical notch. Penis well sclerotized ( Figure 2I), elongate and curved, with membranous apex, and with basal capsule weakly developed. Female unknown.
shape). Moreover, sternite IX of Madeirodula gen. nov. bears paired apophyses, two rod-like sclerites joined apically in the form of an inverted V-shape ( Figure 2D). In other Microweiseinae, there are also paired apophyses, but clearly form an inverted Y-shape (with a single branch in apical part and forked at the base [14]), while in most members of the subfamily Coccinellinae, a single rod-like apophysis is present, or sometimes it is completely reduced [23,24]. The structure of apophyses of the male genital segment in Madeirodula gen. nov. is similar to the condition present in some handsome fungus beetles (Endomychidae sensu lato) [25], and can be regarded as a more primitive form. The asymmetrical tegmen in Madeirodula gen. nov. is of a typical form for the members of Microweiseinae, however, strongly reduced penis guide as well as fused parameres with only a small apical notch separating them is unusual and support distinct evolutionary trends in this lineage. In most remaining Microweisenae, penis guide is elongate with parameres separated and of equal length [8,14], or even completely reduced with only penis guide present, like in Carinodulini [26,27]. Possessing a mixture of morphological characters present in both Carinodulini and Microweiseini, Madeirodula gen. nov. can be considered an intermediate taxon between both tribes. On the other hand, the unique structure of the male terminalia shows more primordial condition than in other tribes of Microweseinae. Further analyses including molecular data have to be conducted to resolve the placement of Madeirodula gen. nov. in the classification of Microweiseinae.
Nothing is known about the biology and ecology of Madeirodula atlantica sp. nov., but large, prominent eyes ( Figures 2F and 3B) and the fact that specimens were collected during the evening when the sun was going down ( Figure 5A,B), may suggest its nocturnal lifestyle.
Members of Microweiseinae are distributed worldwide, with most species occurring in tropical and subtropical regions. Both Serangiini and Microweiseini are distributed pantropically. Carinodulini shows a very interesting distribution pattern with species known from isolated, relictual, mountainous areas in Africa, Asia, and Central and North America. Madeirodulini, discovered on Madeira Island, seemed to 'match' this interesting scattered pattern of distribution ( Figure 5C). However, without thorough investigation of the historical biogeography of the Microweiseinae, it is hard to understand what processes shaped the present distribution pattern of the subfamily, and especially of Carinodulini + Madeirodulini lineage.

Conclusions
The recent field collecting on the Portuguese island of Madeira in the North Atlantic Ocean resulted in the discovery of a new genus and species belonging to the ladybird beetle subfamily Microweiseinae.
Microweiseinae, unlike most coccinellids, have cryptic coloration and a very small body size, rather resembling members of other Coccinelloidea families (Anamorphidae, Eupsilobiidae, or Corylophidae) than 'true' lady beetles. They inhabit mainly leaf litter or under bark habitats and are regarded as a more primitive group of Coccinellidae. Despite several recent taxonomical studies of this group, its diversity and distribution is still largely unknown. The first member of Microweiseinae found in the southwestern ends of the Palaearctic region is described here as Madeirodula atlantica gen. et sp. nov. Its morphology, being a mixture of morphological characters from all known tribes, was the basis for conducting the phylogenetic analysis to test the systematic position of the new genus within the subfamily.
Phylogenetic analysis recovered Madeirodula as a distinct evolutionary lineage within the subfamily Microweiseinae, proposed here as Madeirodulini trib. nov., sister to Carinodulini. Besides the shared morphological characters, both tribes displayed an unusual, very interesting "scattered" pattern of distribution, inhabiting isolated mountainous areas in tropical and subtropical regions around the world. This pattern of the present distribution does not allow, however, to make any conclusions regarding a possible area of origin of the ancestor of Carinodulini + Madeirodulini lineage and subsequent ways of its diversification. Further taxonomical efforts and comprehensive study of the historical biogeography of the entire subfamily Microweiseinae is needed to shed more light into the evolution of this group of lady beetles that have a cryptic appearance and mode of life.