Genetic and taxonomic assessment of the widespread Afrotropical ambrosia beetle Xyleborus principalis (Coleoptera, Scolytinae)

The taxonomy of the widespread Afrotropical ambrosia beetle Xyleborus principalis Eichhoff, 1878 is reviewed based on morphological and molecular data. The species is both morphologically and genetically variable but without any structure corresponding to geography, or between genes and morphology. Examination of type material and other specimens collected across Africa and Madagascar documented the existence of a single species. New synonymies are proposed for Xyleborus principalis (=X. alluaudi Schaufuss, 1897, =X. camerunus Hagedorn, 1910, =X. consobrinus Eggers, 1932, =X. discrepans Schedl, 1950, =X. annectens Schedl, 1957, =X. peramploides Schedl, 1957).


Introduction
Xyleborus Eichhoff, 1864 is a species rich genus of ambrosia beetles found in humid tropical rain forests around the globe. Species in this genus are frequently collected in dead wood or ethanol baited traps and largely dominate wood decomposing beetle guilds in such places (Beaver and Loyttyniemi 1991;Beeson 1961;Schedl 1956;Schedl 1959;Schedl 1977;Thunes 1998). Some of them have very broad distributions and are known to have extended their current circumtropical ranges independently from human transportation (Gohli et al. 2016). Xyleborus species are in principle well adapted to colonize new areas and new introductions to Europe and America are therefore frequently reported (Haack 2001;Haack and Rabaglia 2013;Kirkendall and Faccoli 2010). Global colonization success and subsequent regional expansion is best understood by a peculiar form of reproduction in which siblings mate and assures insemination before dispersal. Permanent inbreeding over evolutionary time scales also makes new colonists resistant to the negative genetic effects of small founding populations (Jordal et al. 2001;Kirkendall et al. 2014).
As one of the more devastating groups of beetles in the timber industry, rapid and accurate identification is important to differentiate serious pest species from less harmful ones, albeit not a trivial task. Many species are indeed very similar to each other, which is a logical consequence of inbreeding without selection for secondary sexual characters often used in species identification. In Africa and Madagascar, one of the most commonly collected species is Xyleborus principalis Eichhoff, 1878, or X. alluaudi Schaufuss, 1897. These two names have been used about each other by various authors, apparently also by the same author. Less frequently used is the name Xyleborus annectens Schedl, 1957, but istogether with a handful of rarely reported taxapart of the same group. The first two species have been reported repeatedly throughout the Afrotropical region from Gambia to Ethiopia and south to South Africa and Madagascar. If one compares collections identified by prominent taxonomists such as Eggers, Schedl, Nunberg, and Browne, it is clear that species diagnoses are weak. A taxonomic revision of the complex is therefore needed where molecular data from multiple genes enable assessment of variation within and between species.  . We tested species boundaries by comparing nucleotide sequences from multiple genes (Tables 1 and 2). DNA was extracted with the Qiagen DNEasy kit, and PCR was made with primers targeting the mitochondrial gene cytochrome oxidase I (COI), the large subunit ribosomal RNA (28S), elongation factor 1-α (EF-1α), and poly-A binding protein 1 (PABP1). We used primers and PCR-cycles described in Mugu et al. (2018). Sequences from four gene fragments were analysed by maximum parsimony both separately and in combination. Because very few substitutions separated nuclear genome sequences in the principalis group and the closest outgroups, we preferred a simple type of analysis as a means to visualize substitutions separating clades and Fig. 1-6 Dorsal, lateral and posterior view of two different Xyleborus principalis morphs. 1-3 narrow morph with less steep declivity and larger spines on interstriae 1 and 3 only, corresponding closely with the X. principalis holotype from Madagascar. 4-6 broader morph with steeper declivity and smaller interstrial granules on interstriae 1-3, corresponding to the holotype of X. annectens from Congo specimens. Node support was estimated by bootstrapping 200 replicates with 100 heuristic searches each, keeping a maximum of 1000 trees to enable searches on matrices with few variable characters.

Materials and methods
Variation in morphology between X. principalis populations and outgroups are described in Table 3. Characters were restricted to the declivity of the elytra (Figs. 3,6) which is the only location on the body that varies within the X. principalis complex. Morphology was studied in a Leica M16 stereoscope. Multiple photographs were taken with a Leica camera on a Leica M205 C stereomicroscope a n d a l i g n e d a n d s t a c k e d u s i n g Z e r e n e S t a c k e r (Zerenesystems.com).

Results
Phylogenetic analysis of morphological characters resulted in a monophyletic X. principalis, with limited support (Fig. 7). This taxon is mainly defined by the curved striae 1 and 2 on declivity, the large-sized strial punctures with a flat bottom, the presence of small and confused punctures on interstriae 1 on declivity, and the short, recumbent strial setae which are no longer than the size of a puncture. Variation within X. principalis was found mainly in five morphological characters (Table 3). There was no geographical pattern in these data, and none of the nodes within X. principalis were strongly supported. Specimens examined varied from having slightly narrower elytra with elevated interstriae on declivity, often with a transverse impression on lower third of declivity, a few large conical granules or spines in interstriae 1 and 3, but with no granules present on interstriae 2 (Figs. 1-3), to the morph which is not impressed on lower third of declivity, has small granules on all interstriae 1-3 on declivity, and is slightly wider and more broadly rounded at elytral apex .
Phylogenetic analyses of nucleotide sequences from four gene fragments resulted in a different tree topology for each gene (Figs. 8-9). The analysis of all molecular data combined resulted in a tree topology identical to the COI topology for X. principalis, and nearly so for the outgroups, with the same nodes supported by near identical bootstrap support.
Xyleborus principalis was monophyletic in all analyses, but without any consistent pattern within this species. The only consistent result was the grouping of four Malagasy specimens in the same clade, but these were mixed with different specimens from the African mainland in the four different gene fragment analyses. Mitochondrial genetic variation was much higher than for the nuclear genes, with a maximum of 14.2% divergence between individuals of X. principalis (Table 4). These same specimens differed only by a single substitution at the 28S gene, and four in total for the three genes combined (see Fig. 9).
Examination of type material and other specimens identified as X. principalis, X. alluaudi, and X. annectens by Schedl, Nunberg, Browne and Eggers revealed no consistent differences (Figs. 1-6). Additionally, the holotypes of X. peramploides, X. consobrinus, and X. discrepans were found to be within the same range of variation. Illustrations of X. alluaudi by Schedl (1977) and Nunberg (1978) correspond to the first and second morphotype, respectively, while Nunbergs illustration of X. principalis fits the first morphotype. The same author illustrates X. annectens in very much the same way as for X. principalis. All three illustrations by Nunberg has a scale indicating exactly 3.0 mm length for all three taxa.

Discussion
Based on the great similarity in morphological characters in six named Afrotropical species of Xyleborus, we conclude that only one species exists -X. principalis. Genetic data indicate high mitochondrial variation within this species, but there is no correlation between morphology and genetic variation. Neither is there a consistent pattern in the variation across genes, and there is no geographical pattern in the genetic or morphological data. Although more than 10% divergence was observed for COI between several subgroups of X. principalis, such high intraspecific variation in the mitochondrial genome is quite normal for permanently inbreeding species (Andersen et al. 2012;Cognato et al. 2019;Gohli et al. 2016;Kambestad et al. 2017). At the same time there is very limited variation in nuclear genes for this species, most notably no more than two substitutions in the 28S gene. Several species in the Hypothenemus eruditus Westwood, 1836 complex, but also in related Xyleborus species such as X. ferrugineus (Fabricius, 1801), harbour higher genetic variation than in X. principalis. It is therefore likely that X. principalis has maintained large genetic variation over time in the same manner as for other permanently inbreeding species, and is reflected in morphological polymorphism.
Karl Schedl had a rather unfortunate habit of describing new species based on tiny differences from the holotype of a known species (Wood and Bright 1992). Many of these erroneous taxa are now synonymized, particularly in Neotropical and Oriental species which have been studied in more detail. We would therefore not be surprised if many new synonyms are proposed as the revision of the African fauna proceeds.  Xyleborus consobrinus Eggers, 1932, n. syn. Xyleborus discrepans Schedl, 1950, n. syn. Xyleborus annectens Schedl, 1957, n. syn. Xyleborus peramploides Schedl, 1957. n. syn.

Revised diagnosis
Length 2.9-3.6 mm, 2.4-2.8 × as long as wide, colour reddish brown. Pronotum smooth and shiny on posterior half, abruptly declivitous and strongly asperate on anterior half. Elytra with striae in regular rows on disc, on declivity punctures larger and more irregularly placed, bottom of puncture flat with margins clearly marked, punctures sometimes contiguous, or well separated, striae 1, 2 and sometimes 3 usually curved laterally; interstriae on disc smooth, on declivity with granules, sometimes missing on interstriae 2, sometimes two or more granules on interstriae 1 and 3 larger than the average size of a granule; punctures in interstriae much smaller than in striae, variable in size, on interstriae 1 small and strongly confused. Vestiture consisting of long and thin interstrial setae, intermixed with, or entirely replaced by, thicker setae; strial setae fine, recumbent, barely longer than diameter of a strial puncture. Elytral apex broadly to more narrowly rounded, sometimes with a distinct transverse impression on lower third, sometimes terminal interstriae 1-2 slightly elevated to make apex more pointed. Protibiae with 6 lateral socketed teeth, metatibiae with 8-10 lateral teeth on distal two-thirds.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.