Ancient diversity of Afrotropical Microborus: three endemic species – not one widespread

Abstract The primarily Neotropical genus Microborus Blandford is represented with three species in Africa and Madagascar. The previously recorded species from this region, M. boops Blandford, is a Neotropical species restricted to Central America and is likely not found in the Afrotropics. The previously recognised species in western parts of Africa is M. camerunus (Eggers) and is resurrected from synonymy under M. boops. Molecular and morphological data revealed a second species of this complex in Madagascar, M. brevisetosus Jordal. Another new species, M. angustus Jordal, co-occurs with M. camerunus in Cameroon. Substantial genetic divergence indicate that Microborus was established in the Afrotropical region long before human transport across oceans. A key to Afrotropical species is provided.


Introduction
Microborus Blandford, 1897 is a largely Neotropical genus consisting of eight known species, with one of these also recorded from the Afrotropical region. Species are generally small in size, but are often taken from very thick bark of large tree trunks (Wood 2007). Their breeding biology is unusual in that nests are initiated via the entrance opening of a much larger bark or ambrosia beetle species and mines away from their host gallery just inside the entrance (Figs 1-2).
Previous classifications have placed Microborus in the scolytine tribe Hexacolini (previously Ctenophorini, see Alonso-Zarazaga and Lyal 2009). Recent molecular phylogenies have nevertheless questioned the relationship to Scolytodes Ferrari and other hexacoline genera, and instead pointing towards a relatively isolated position in Scolytinae. This is a very old genus that apparently diverged from all other extant lineages more than 100 Ma (Jordal and Cognato 2012) and which experienced very little morphological change since its time of origin (Cognato and Grimaldi 2009).
The supposedly broad distribution of Microborus boops Blandford in the Neotropical and Afrotropical regions has been inferred as a recent introduction to Africa and Madagascar (Wood 1982). Using integrated morphological and molecular data, the Afrotropical fauna is revised, including discovery of two new species, and rejection of a globally widespread distribution in M. boops.

Materials and methods
Specimens included were collected during the author's field expeditions to Cameroon (2007) and Madagascar (2015). Deposition of typematerial are indicated by the following acronyms: BMNH, Natural History Museum London; CAS, California Academy of Science; NHMW, Naturhistorisches Museum Wien; ZMBN, University Museum of Bergen (formerly Zoological Museum, Bergen). DNA was extracted from whole specimens, of which the macerated body remains were mounted on slides or glued on a pinned card. Six gene fragments were amplified: COI, EF-1α, 28S, CAD, ArgK and PABP1 (Jordal et al. 2011;Pistone et al. 2016). Sequences were concatenated for combined phylogenetic analyses using maximum likelihood and maximum parsimony in PAUP* (Swofford 2002).
Morphological examination of internal or hidden characters such as flight wings, proventriculus and male genitalia was only made for one species that had sufficient specimens available.

Microborus angustus
Diagnosis. A very elongated, almost black species, with impressed elytral striae and a distinct costate rim along the postero-lateral margin of elytral declivity.
Head. Eyes separated above by 1.4 × their width. Frons reticulate and deeply punctured, smooth and shiny at level of antennal insertion, vestiture consisting of a few scant fine setae.
Legs. Protibiae with three lateral teeth (embedded denticles), and one additional tooth just above the inner mucro; posterior face smooth.
Ventral vestiture simple, on ventrites very fine, short setae. Wings typical for weevils, costa with two setae close to each other near base, and one seta two-thirds the distance towards the stigmal patch; anal field missing, posterior margin with long fine setae; stigmal patch with two short, sharp setae, each on a small tubercle.
Proventriculus with apical plate well developed, median suture wide open, sutural teeth long and sharp, apical teeth and marginal bristles missing, closing teeth long and prominent, >10 large femoral teeth.
Male genitalia very simple, spiculum gastrale not present, no distinction between apophyses and aedeagal body, internal sac with granulated surface, tegmen open dorsally, gradually broader ventrally with a short manubrium.
Etymology. Latin adjective angustus, meaning narrow. This is the most elongated species in the genus in the Afrotropical region.
Distribution and biology. Only known from the type locality. It was taken together with M. camerunus (Eggers, 1919) under thick bark of a fallen Ficus tree. Both species used entrance holes made by Xyleborus principalis Eichhoff, 1878.  Description (male and female). Length 1.4 mm, 2.6 × longer than wide. Colour reddish brown, pronotum darker.
Head. Eyes separated above by 0.6 × their width. Frons reticulated and lightly punctured, protruding slightly below eyes, vestiture consisting of >50 short setae, longer between eyes and some on epistoma.
Pronotum smooth, shiny, with densely placed puntures. Elytra with striae impressed, punctures deep, spaced by distance equal to their diameter; interstriae about half as broad as striae, with very fine irregularly spaced punctures; postero-lateral interstrial rim slightly elevated with 2-3 blunt granules. Vestiture consisting of a few longer, erect, golden setae on discal interstriae, with densely placed, short, stiff setae on declivity.
Legs. Protibiae with three lateral teeth (embedded denticles), and one additional tooth just above the inner mucro; posterior face rough.
Etymology. Latin adjectives brevis, meaning short, and setosus, meaning bristly, referring to the very short stiff interstrial setae on the elytral declivity.
Distribution and biology. Madagascar: Boeny, Melaky, Diana and Analanjirofo provinces. Specimens were examined only from the western part of the island. It is presumed that Schedl's reported specimens from the east and north of the island are conspecific. The collection from Ankarafantsika ( Fig. 1) (Eggers, 1919): synonymized with M. boops Blandford, 1897, by Wood (1982, here resurrected.

Type material examined. Holotype of Pseudocrypturgus camerunus Eggers (NHMW). Holotype of Microborus boops Blandford (BMNH).
Diagnosis. Distinguished from M. brevisetosus and M. boops by the smooth and glabrous frons, the glabrous central area of the ventrites, the smooth posterior face of the protibiae, subconfluent strial punctures, and the slightly stouter body shape.
Head. Eyes separated above by 0.7 × their width. Frons smooth, shiny and lightly punctured, vestiture consisting of <10 short setae on epistoma and 2 longer setae between eyes.
Legs. Protibiae with three lateral teeth (embedded denticles), and one additional tooth just above the inner mucro; posterior face smooth.
Ventral vestiture simple, on ventrites consisting of a few irregularly placed short setae close to the lateral margins.

Molecular data on Afrotropical species
Gene sequences obtained via PCR are listed by their genbank accession numbers in Table 1. Maximum likelihood and maximum parsimony provided consistent results across analyses, with all nodes maximally supported (Fig. 21) about each species validity. The largest nuclear variation was found in 28S (3.9%), a substantial difference for morphologically similar taxa (see e.g. Jordal and Kambestad 2014). Guided by the molecular data, a search for consistent morphological differences was found in the frons, elytral declivity and the venter of these beetles. Hence, the overall similarity that has led previous researchers to synonymise M. camerunus with M. boops (Wood 1982), emphasizes the need for careful consideration of possible semi-cryptic character differences. The low rate of change in morphological characters for the genus as a whole, as documented by the close similarity to the mid-Cretaceous fossil M. inertus Grimaldi, 2009 (see Cognato andGrimaldi 2009), makes it advisable to base new synonymies on genetic data and rigorous morphological examination.