Sky islands of the Cameroon Volcanic Line support the westernmost clade of five new Typoderus weevils (Coleoptera: Curculionidae: Molytinae)

The weevil genus Typoderus is for the first time reported west of the Congo basin. Analysis of 2,136 aligned positions from one mitochondrial and two nuclear fragments revealed a moderately supported clade of five new Cameroonian species: T. amphion sp. nov. (Mt. Oku), T. canthus sp. nov. (Mt. Oku), T. clytius sp. nov. (Mt. Cameroon), T. iphitus sp. nov. (Mt. Kupe) and T. telamon sp. nov. (Mt. Kupe). Molecular clock analysis of 20 DNA barcode fragments using a fixed substitution rate estimated divergences within this clade to be during the Middle to Late Miocene (10.5–5.4 million years ago, MYA), which pre-dates the onset of the Pliocene-Pleistocene global climatic fluctuations and corresponding cycles of African forest size fluctuation. Such relatively old dates are unexpected and might reflect four unavoidable shortcomings of the temporal analysis: 1. undersampled ingroup, 2. scarcity of comparative temporal data for other animal clades from the Cameroon Volcanic Line, 3. oversimplification of a fixed-rate molecular clock approach using a single maternally-inherited protein-coding marker and 4. possible overestimation of comparatively old ages when using largely saturated mitochondrial sequences. Two obscure weevil species from the Republic of the Congo are hypothesized to belong to the genus Typoderus: T. distinctus (Hoffmann, 1968) comb. nov. (from Anchonidium subgenus Neoanchonidium) and T. baloghi (Hoffmann, 1968) comb. nov. (from Anchonidium subgenus Subanchonidium). Three genus-group names are newly synonymized under Typoderus: Entypoderus Voss, 1965 syn. nov. (the only non-nominative subgenus of Typoderus), Neoanchonidium Hoffmann, 1968 syn. nov. (subgenus of Anchonidium) and Subanchonidium Hoffmann, 1968 syn. nov. (subgenus of Anchonidium). Habitus images and other supplementary information of all sequenced specimens are available online at dx.doi.org/10.5883/DS-VGDS005 and dx.doi.org/10.5883/DS-VGDS006.


Introduction
This paper was triggered by a discovery of unexpected weevils in three Cameroonian localities: Mt. Oku, Mt. Cameroon and Mt. Kupe. When first seen, the specimens appeared to belong to five species of the flightless forest-dwelling weevil genus Typoderus Marshall, 1953. A peculiarity of these finds was that the genus had never been recorded west of the Congo basin. For most of its history Typoderus contained 11 nominal Afrotropical species each known only from the type series. Grebennikov (2017) demonstrated monophyly of the genus and hypothesized Lupangus Grebennikov, 2017 as its sister group. Presently the genus Typoderus consists of 14 flightless species restricted to wet Afrotropical forests (Fig. 1). Eight Typoderus species each known only from the type series were described from Rwanda, the Democratic Republic of the Congo and Angola. The remaining and better known six species inhabit the Tanzanian forest archipelago (sensu Grebennikov and Heiss 2018, i.e. isolated sky island forests of different age separated by vast stretches of hot and dry savannah) and were recently studied using DNA data and phylogenetic methodology (Grebennikov 2019b).
Remarkably, the genus Typoderus has never been reported throughout most of the Congo basin and along its western rim. Such an askew distribution of this genus (and of other forest-dependent weevils such as Allocycloteres Voss, 1965, Paocryptorrhinus Voss, 1965, Apa ro pionella Hustache, 1939, Prothrombosternus Voss, 1965, or Tazarcus Grebennikov 2020; see Voss 1965, Gre bennikov 2015 respectively) might be an artifact of insufficient sampling and/or inadequate and misleading taxonomy. This was suggested by the fact that representatives of many genera that were originally reported from one side of the Congo basin, were eventually detected on the other side. Recent examples include Xenocaucus China and Usinger, 1949 assassin bugs (Weirauch et al. 2017), Coelocorynus Kolbe, 1895 Trichiini scarab beetles (Šípek et al. 2009), soft-bodied Dexoris Waterhouse, 1878 net-winged beetles (Bocak et al. 2013) and a clade of two likely synonymous genera of pill scarabs (Afrocloetus Petrovitz, 1968and Congomostes Paulian, 1968: Grebennikov 2019c. The first hint that Typoderus might be present on the western side of the Congo basin came when attempting to elucidate the identity of two obscure species, each of them the type species of an equally obscure monotypic subgenus: Anchonidium (Neoanchonidium) distinctum Hoffmann, 1968 and A. (Subanchonidium) baloghi Hoffmann, 1968 from the Republic of the Congo (Fig. 1). The only available evidence was a dorsal image of one of the holotypes ( Fig. 1) exhibiting all morphological synapomorphies of Typoderus. This hypothesis could not be adequately tested, however, since the type series remain unavailable for study (see below), and both species have never been re-sampled.
The second and more convincing indication of Typoderus west of the Congo basin was the aforementioned detection of Typoderus-like weevils in the Cameroonian highland forests. Adults of these beetles displayed the diagnostic feature uniquely distinguishing the Typoderus + Lupangus clade among all Afrotropical weevils: pronotum on each side with two longitudinal ridges, of which the inner ridge is twice bent to form a zig-zag (Fig. 1,  fig. 2G in Grebennikov 2018). Additionally, all Cameroon ian specimens were distinguishable from those of Lupangus by two more Typoderus-only characters: (2) eyes not more than 2.5x as high as wide and (3) lack of a deep transverse groove on head immediately behind the posterior margin of eye (both characters illustrated for Lupangus in fig. 4, Grebennikov 2017). Notably, two morphologically distinct groups of such specimens were collected on both Mt. Oku and Mt. Kupe suggesting two cases of sympatry, while only one morphospecies was seen on Mt. Cameroon. These Cameroonian weevils were distinct from those living in Tanzania (Fig. 3) and likely belonged to unnamed species.
The three forested Cameroonian highlands supporting the suspect Typoderus beetles are remarkable in their own right. They are parts of the Cameroon Volcanic Line (CVL, Déruelle et al. 2012) formed by a chain of (mainly extinct) volcanoes extending for about 1,700 kilometres between the island of Annobón in the southwest and Lake Chad in the north-east. Besides the prominent continental part including all three aforementioned highlands, the oceanic part of CVL includes Pico de São Tomé (2,024 metres), Pico do Príncipe (947 m) and Pico Basilé (3,011m). Similarly to the Albertine Rift located between Lake Albert and Lake Tanganyika and the Eastern Arc Mountains in mainly Tanzania, CVL supports exceedingly diverse life forms, many of them restricted to a single highland. The Albertine Rift has the highest vertebrate diversity in Africa (Voelker et al. 2010), while both Cameroon and Tanzania have the highest per-country chameleon diversity on the continent (Tilbury 2018). This extraordinary high biodiversity is normally linked with the availability of the elevational landscape gradient formed by these archipelago-type chains of highlands. Dated at about 30 million years (MY, Burke 2001;Reusch et al. 2010), the continental part of CVL is sufficiently old enough to serve as climatic refugia for moisture-dependent lowland organisms when climate change forces them to retreat into cooler and/or wetter highlands. This was particularly important during multiple repeated warm and dry interglacials of the Pliocene-Pleistocene (Plana 2004), when Afrotropical rainforest recurrently shrank to about 10% of its present size ( fig. 4 in Hamilton and Taylor 1991), leaving these highlands to form sky islands of forest surrounded by vast stretches of dry and hostile savannah. The resulting extended and repeated isolation of forest biota left behind by the retreating lowland Congo basin forest supposedly drove speciation in all three chains of highlands. When tested for endemic CVL clades, this spatio-temporal hypothesis was corroborated by post-Miocene divergence dates of two species of Otomys Cuvier, 1824 laminate-toothed rats (Taylor et al. 2014), although in Phrynobatrachus Günther, 1862 puddle frogs, the timing of diversification predates the Plio-Pleistocene (Zimkus and Gvoždík 2013, see also Discussion).
Three phylogeographic processes are commonly evoked to explain the high diversity and spotty distribution of Afrotropical sky island biota. Some of these organisms are considered species-poor paleoendemics retained from the deep (=pre-Pliocene) past and sisters to sizable radiations (Fjeldså and Lovett 1997;Grebennikov 2019a). More speciose clades with more recent diversifications are thought to be products of the Plio-Pleistocene vicariant speciation achieved through habitat fragmentation (Grebennikov 2019c) and additionally enriched via habitat re-connection and secondary sympatry of recently divergent clades (=the "species-pump" hypothesis, Papadopoulou and Knowles 2015). It was, therefore, intriguing, to test, to the extent possible, each of these three scenarios using the discovered CVL beetles and by doing this, to shed light on their taxonomy and the evolutionary past.
The purpose of this paper is to document analytical steps triggered by the discovery of suspect Typoderus weevils west of the Congo basin. Working within the logical framework of testing falsifiable hypotheses (Popper 1959), the following questions (=hypotheses H1 to H5) were tested by performing phylogenetic analyses of DNA sequence data: Hypothesis 1 (H1): all five visually recognized and geographically structured morphospecies of CVL Typo deruslike weevils correspond to biological species (following the unified species concept, De Queiroz 2007); Hypothesis 2 (H2): these beetles taxonomically belong to the genus Typoderus; Hypothesis 3 (H3): at least one of the newly discovered CVL candidate species represents a paleoendemic; Hypothesis 4 (H4): at least one divergence between allopatric CVL candidate species is attributable to simple vicariance via habitat isolation of CVL sky islands during the post-Miocene climatic fluctuations; Hypothesis 5 (H5): at least one case of sympatry of CVL candidate species is attributable to a secondary meeting of recently speciated populations through temporary habitat reconnection (the "species-pump" hypothesis).
Last but not least, an attempt is made to fine-tune taxonomy pertaining to the relevant parts of the weevil Tree of Life by revising rank-based names and making them to reflect the best available phylogenetic hypothesis.

Specimen sampling, analyses design and DNA sequencing
All newly reported specimens of CVL Typoderus-like beetles are adults obtained from fifteen forest litter samples (Table 1) in wet primary forests of Mt. Oku, Mt. Cameroon and Mt. Kupe in Cameroon using standard sifting methods (Grebennikov 2017(Grebennikov , 2019c. This paper is a sequel to the recent work focused on Tanzanian Typoderus (Grebennikov 2019c) and the sister clade of the genus (Grebennikov 2017), therefore it re-uses already reported DNA data and re-employs the same laboratory and analytical procedures. In brief, about a hundred newly sampled CVL Typoderus-like weevils were preliminary sorted into five geographically-and visually-coherent morphospecies. External diagnostic morphological characters for these morphospecies are given in Table 4 and were clear-cut and diagnostic for all individual specimens examined (except for two characters with intraspe-   (Table 3). Additionally, 14 of them were sequenced for two more markers, in support of analysis A3 (see below; Table 3).
To test hypotheses H1-H5, the total of four DNA analyses was implemented.
First analysis (A1) designed to assess hypothesis H1 (five morphospecies represent five biological species) by testing whether DNA barcode clusters match morphological and geographical grouping of specimens. By doing so, consistent signal was sought from different and independent sources of evidence in an attempt to delimit independent evolutionary units herein described as new species. For this purpose, 20 newly generated DNA barcodes 531-658 bp in length were analysed using the Neighbour Joining (NJ) method, Barcode Index Number cluster identification algorithm (BIN; Ratnasingham and Hebert 2013) and Kimura 2 parameter, as implemented in the online engine of the Barcode of Life online database (=BOLD, Ratnasingham and Hebert 2007, http://www. boldsystems.org).
To estimate evolutionary divergence over sequence pairs for groups recognized in section 4.2 below as five new Cameroonian Typoderus species, 14 terminals representing these species were analysed separately using MEGA5 (Tamura et al. 2011). The 2136 position matrix was reduced to that of a total of 1599 positions, by removing positions containing gaps and missing data. Two pairs of values were calculated: the number of base differences per site from averaging over all sequence pairs within and between species, as well as uncorrected p-distance.

Second analysis (A2): designed to test hypothesis H2
(CVL weevils taxonomically belong to the genus Typoderus). Considering (1.) diagnostic morphological characteristics of CVL specimens strongly in support of this hypothesis, (2.) lack of a clearly identified sister-group of the clade formed by Typoderus and its species-poor sister group of the genus Lupangus; (3.) multiple non-monophyly of Molytinae (Shin et al. 2017) and (4.) the lack of new significant DNA data on Molytinae (as compared to those recently released, Grebennikov 2017), a full-sized phylogenetic analysis with multiple non-Typoderus outgroups was not deemed feasible or, indeed, necessary. Instead, all 20 newly generated DNA barcodes were identified by using phenetic methods such as the BLAST algorithm implemented in GenBank (Altschul et al. 1997) or through the Neighbour Joining (NJ) clustering method (as in Analysis A1). This comparison was done against all Curculionidae records currently in BOLD (5,117 species with barcodes, 55,115 specimens with barcodes; as on November 25, 2020).

Third (topological) analysis (A3): designed to shed
light on all five hypotheses. Fourteen CVL specimens representing all five morphospecies (as recovered on the NJ tree in analysis A1) were additionally sequenced for two nuclear ribosomal loci: internal ribosomal spacer 2 (ITS2) and 28S rDNA ( Table 2). All corresponding laboratory work was done in CCDB using protocols and primers described earlier (Grebennikov 2017). The three-marker dataset was formed by 14 newly sequenced CVL terminals (the ingroup) merged with the 71 terminal dataset from Grebennikov (2019b); the latter containing 70 Tanzanian Typoderus (the outgroup) and a single Lupangus terminal to root the topology. Images of all 85 sequenced specimens and their DNA data are available online at BOLD dataset dx.doi.org/10.5883/ DS-VGDS005. Alignment of the ITS2 and 28S sequences was made using the MAFFT 7 online platform (http:// mafft.cbrc.jp/alignment/server) and the Q-INS-i algorithm utilising the secondary structure information (Kuraku et al. 2013;Katoh et al. 2017). To correct for visually detected alignment errors, 34 positions at the 5 end of four relatively short ITS2 sequences were manually realigned (specimens 2130: 10 positions; 2154: eight positions; 3094: three positions and 7164: 11 positions). No other modifications to the alignments were made and no parts of the alignments were excluded from the analysis. The resulting matrix of 85 terminals had 2,136 aligned positions (Table 2) and 23% missing data (mainly due to insertions and deletions in ITS2). GenBank accession numbers of new DNA sequences from 14 CVL weevils are in Table 3. The concatenated matrix was partitioned into three fragments (Table 2) and an independent CAT approximation (Stamataikis et al. 2008) of the GTR+G model was applied independently to each data partition (Abadi et al. 2019). Phylogenetic analysis was conducted on the CIPRES Science Gateway online platform (Miller et al. 2010; http://www.phylo.org) using the Maximum Likelihood (ML) method and RAxML 8 tool (Stamatakis 2014). Support values were obtained with 1000 bootstrap replicates (Stamatakis et al. 2008) and topology was visualized in FigTree v1.4.0 (Rambaut 2019).
Fourth (temporal) analysis (A4) was used to partially test hypotheses H3-H5, each of them requiring dates on species divergences. Considering monophyly of CVL Typoderus-like weevils (see Results) and the lack of relevant calibrating points, a fixed-rate temporal analysis was the only available option. For this purpose the matrix from the analysis A1 consisting of 20 DNA barcodes of CVL weevils was re-analysed and no outgroup used. To estimate divergence times, a fixed molecular clock rate of 0.018 nucleotide substitutions per site per million years per lineage (subs/s/MY/l) was applied. This value is consistent with those obtained in other beetles (Papadopoulou et al. 2010;Andújar et al. 2012), other insects (Brower 1994) and other arthropods (Bauzà-Ribot et al. 2012), and was used for dating evolutionary events in Tanzanian Typoderus (Grebennikov 2019b). Bayesian phylogenetic analysis in BEAST 1.8 (Drummond et al. 2012) was used to simultaneously estimate an ultrametric phylogenetic tree and rates of diversification. The HKY+G evolutionary model (estimated in MEGA 7, Kumar et al. 2016) was applied and the MCMC chains ran for 10 million generations. Convergence of all parameters was checked in TRACER (Drummond et al. 2012) and consensus trees were estimated with TreeAnnotator (Drummond et al. 2012) discarding 25% initial trees as a burn-in fraction. No a priori topological constrains (such as rooting) were added in the analysis.

Species diagnosis and description
Various species description workflows (Riedel et al. 2013;Meierotto et al. 2019) converged on importance of providing (1.) DNA barcode, (2.) precise locality data and (3.) image of the holotype. Here I follow the new species description workflow adopted for the Typoderus + Lupangus clade (Grebennikov 2017(Grebennikov , 2019b) by presenting species-level diagnostic morphological characters in a table (Table 4). These characters consistently list all easily observed differences of five new species, including those of male genitalia. The latter were dissected only for holotype male specimens (distinguished from females by slightly depressed abdominal ventrites 1 and 2).
No attempt was made to compare male genitalia within the newly described species, nor to dissect and study female genitalia. Considering that Typoderus form narrow-range morphologically and genetically distinct clades in at least relatively well-sampled Tanzania (Grebennikov 2019b), no consistent attempt was made to compare morphology of five herein described new Cameroonian species with that of six Typoderus from Tanzania (Grebennikov 2019b). As before, the holotype body length (measured in dorsal view between anterior margin of pronotum and apex of elytra) is the only reported measurement; all other measurements and ratios can be obtained from images (dx.doi.org/10.5883/DS-VGDS006). Overall, taxonomic procedures implemented in this paper were designed to consistently generate valid species names with the least effort, with focus on diagnosis, not descriptions (Wheeler et al. 2012;Renner 2016;Miralles et al. 2020;Vences 2020). This approach termed "turbo-taxonomy" (Butcher et al. 2012;Summers et al. 2014) or fast-track taxonomy (Riedel et al. 2013) was designed to expedite naming of new species in particularly understudied and species-rich clades, which the genus Typoderus appears to be. Other examples include Neotropical Gracillariidae leaf-mining moths (Leed et al. 2014) or Cecidomyiidae gall midges; the latter cosmopolitan family of some 6-7 thousand species was estimated to contain up to 1,800,000 species, a bewildering number exceeding that of the total of all scientifically named animal species (Hebert et al. 2017).

Results
The first analysis (A1) clustered all 20 DNA barcodes of CVL weevils fully in agreement with their preliminary grouping in five geographical morphospecies (Fig. 2). The latter form exclusive clusters each. Four CVL morphospecies each correspond to a single BIN (ACT0016, ACT2982, ADJ8842, ADK5949), and one morphospecies was split into two BINs (ADK6062, ADM2013). The sequence data of the barcodes was nearly complete  The second analysis (A2) had all 20 DNA barcodes of CVL weevils consistently matched (through BLAST comparison or using BOLD identification) with those of the genus Typoderus.
The third analysis (A3) using the ML phylogenetic method and a concatenated 2,136 bp matrix of 85 terminals produced a well-resolved tree (Fig. 3) topologically identical to the one in Grebennikov (2019b), except for  (Table 3) and sifting sample number (Table 1). Habitus images are to scale; their numbers indicate exact Cameroonian specimens imaged. Names next to clades are those of the new species described in section 4.2.
the addition of the CVL terminals. All 14 Cameroonian terminals formed a moderately supported clade (bootstrap support 75%) sister to a strongly supported clade of T. peleus Grebennikov, 2019 and T. antennarius Voss, 1965 (Fig. 3). All five CVL morphospecies formed strongly supported clades (each 100%). Four CVL species (expect the one from Mt. Cameroon) formed a strongly supported clade (100%); its internal resolution was moderately supported, with a Mt. Kupe species sister to a clade (77%) consisting of a species from Mt. Oku, itself sister to two species from Mt. Oku and Mt. Kupe (68%, Fig. 3).
The fourth analysis (A4), estimating divergence dates of 20 CVL DNA barcodes, resulted in a topology (Fig. 4) identical to those recovered by analyses A1 and A3 (Figs. 2 and 3, respectively). All divergences of the Cameroonian clade were estimated to take place in the Middle to Late Miocene (Fig. 4). The most recent common ancestor of this clade lived 10.5 MYA (95% credibility interval 14.6-7.5 MYA), while the most recent species-level divergence took place at 5.4 MYA (3.9-7.1 MYA).

Hypotheses
Interpretation of the herein presented results led to conclude that: Hypothesis 1 (five preliminary recognized and geographically coherent morphospecies of CVL Typoderus-like weevils are biological species) is strongly supported, since three independent lines of evidence (morphology, geography, DNA) groups specimens in identical clusters herein assumed to be discrete biological species. The sympatric occurrence of two strongly genetically diver-gent pairs (at Mt. Oku and at Mt. Kupe) offers an additional argument for species-level distinctness of these lineages. Moreover, the herein defined species (including both sympatric pairs) exhibit constant differences not only in COI (Fig. 2, a mitochondrial marker), but also in comparably rapidly evolving ITS2 (while having almost no differences in more conservative 28S; both are nuclear markers). Such concordance of unlinked markers (mtD-NA vs. nucDNA) offers further evidence for species-level distinctness. The species to be named T. iphitus in section 4.2 might consist of two cryptic species due to the deep barcode divergence in the same order as that between the other species, but this needs to be investigated in more detail and is beyond the scope of this paper Hypothesis 2 (all analysed CVL weevils belong to the genus Typoderus) is strongly supported, since phenetic similarities in DNA sequences (analysis A2) reinforce morphological identifications and assign all specimens to monophyletic Typoderus. Consequently, these CVL weevils are consistently referred to as Typoderus using five new species-group names introduced below.

Hypothesis 3 (at least one of the newly discovered CVL
Typoderus is a paleoendemic) is weakly rejected, since none of CVL Typoderus forms a sister-group to a well-supported clade containing non-CVL species and reliably well-known and sufficiently old divergence date (although the cut-off date can hardly be precisely defined).
Hypothesis 4 (at least one divergence between allopatric CVL Typoderus species might be attributed to simple vicariance via habitat isolation of CVL sky islands during the post-Miocene climatic fluctuations) is weakly rejected, because by the beginning of the Pliocene at 5.3 MYA all five species of CVL Typoderus have already diverged (Fig. 4, but see below on the limitations of the temporal analysis). . Maximum clade credibility Cameroonian Typoderus tree with median ages derived from the BEAST analysis A4. The 95% credibility interval for each node is given with a horizontal bar. Digits at nodes are million years before present. Terminal names consist of specimen number (Table 3), GenBank accession number and sifting sample number (Table 1). Names next to clades are those of the new species described in section 4.2.
Hypothesis 5 (at least one case of sympatry of CVL Typoderus might be attributed to a secondarily meeting of recently spectated populations through temporary habitat reconnection; the "species-pump" hypothesis) is weakly supported. In both cases of CVL sympatry (T. amphion sp. nov. and T. canthus sp. nov. co-occurring on Mt. Oku and T. iphitus sp. nov. and T. telamon sp. nov. co-occurring on Mt. Kupe) sister clades of each of four species occur on a different, although a relatively nearby CVL sky-island, which might have served as a source of secondary colonisation.
As a word of caution, the herein undertaken attempt to study spatial and temporal aspects of CVL Typoderus evolution is subject to four significant limitations. Firstly, the genus remains acutely undersampled. No DNA data on Typoderus are available from the >3,000 km gap separating sequenced populations in Tanzania and Cameroon, and even these two best studied countries likely have just a fraction of their evolutionary distinct Typoderus lineages samples and studied. It seems unlikely that Typoderus is truly absent from most of the Congo Basin lowland rainforest (Fig. 1), since focused litter sampling in nearby Tanzania detected this genus in all but two targeted forest blocks (Grebennikov and Heiss 2018; those two are geologically recent volcanoes Mt. Hanang and Mt. Meru likely not yet colonized by the genus). When available, new data are expected to modify, or perhaps even negate, some of the herein presented conclusions (for example, detection of non-monophyly of CVL Typoderus or perhaps significantly different divergence dates).
Secondly, very few other CVL animal clades were studied in sufficient detail to permit meaningful comparison. Similar to Typoderus weevils, all CVL laminate-toothed Otomys rats are monophyletic (two species diverging in the Pleistocene) and represent the western-most records of the genus, which is absent throughout most of the lowland Congo Basin ( fig. 1 in Taylor et al. 2014). Phrynobatrachus puddle frogs widely distributed in sub-Saharan Africa are also represented in CVL by an endemic clade only and, similar to Typoderus, the clade's divergences took place in Middle to Late Miocene (Zimkus and Gvoždík 2013; the analysis was perhaps biased to older dates by using suboptimal priors, email correspondence with V. Gvoždík on August 12, 2019). These two papers are the only ones providing phylogeographic results comparable with other of CVL Typoderus.
Thirdly, and perhaps most significantly, the herein implemented temporal analysis utilizes a fixed-rate molecular clock of a single maternally-inherited protein-coding marker. This methodological oversimplification is unavoidable, since no other calibrating methods are presently available for this understudied clade. This substitution rate agrees with those of other Arthropoda (see four references in Material and Methods), however its application to CVL Typoderus might, or might not, be correct, because at least a four times greater rate has been detected in the same DNA fragment among similarly wingless Trigonopterus Fauvel, 1862 weevils from the Sunda Arc (0.0793 subs/s/MY/l, analysis 2 in Tänzler et al. 2016, versus 0.018 subs/s/MY/l implemented herein).
If the CVL Typoderus rate is at least twice higher than the one implemented in analysis A4, then the origin of the CVL clade's crown group and its diversification would be estimated at the beginning of the Pliocene (Fig. 4). Such a result would reverse the present rejection of hypothesis H4, and suggest a link between CVL Typoderus evolution and post-Miocene cyclic climatic fluctuations.
Fourthly, the herein implementer temporal analysis possibly overestimated the ages, necessarily using (largely saturated) mitochondrial sequences to estimate comparatively old ages (as demonstrated Near et al. 2017 for ray-finned fishes). Without relevant fossil evidence and improved phylogenetic resolution within the Molytinae, it is not possible to fine-tune the herein implemented temporal analysis A4 and, therefore, these results should be taken with caution.
Notwithstanding these limitations, the most significant phylogeographic result of this study is that all Typoderus weevils currently known west of the Congo basin form a clade (Fig. 3). This, together with the fact that the sister group of Typoderus is strictly Tanzanian in distribution, seemingly suggests an East African origin of Typoderus and single colonization of Cameroon. However, with such little DNA data available for the, likely, many dozens (if not hundreds or perhaps thousands) of other Typoderus species, this cannot be concluded with sufficient confidence. Marshall, 1953: 104. Type species: Typoderus machadoi Marshall, 1953 by original designation. = Entypoderus Voss, 1965 syn. nov. Voss 1965: 332 (as subgenus of Typoderus Marshall, 1953. Type species: Typoderus deceptor Marshall, 1953 by original designation.

Typoderus Marshall, 1953
Available evidence. Voss (1965: 332) established Entypoderus for all Typoderus having seven antennomeres in the funicle and contrasted it with the nominative subgenus with five antennomeres in the funicle. In this situation, use of a single diagnostic character necessitates that at least one of its two states is not synapomorphic and, therefore, the proposed classification is not phylogenetic. Moreover, no practical need is served by sustaining subgenera in Typoderus, a relatively small genus of only 19 species (inclusion five new one and two new combinations; see below). Perhaps the entire category of a subgenus (as well as a subspecies; see ZINK 2004) might be entirely abolished in zoological classification, since its practical advantage as a name is often compromised by the unwieldiness of non-binominal species-level nomenclature. In other words, trinomial (or even tetranomial, if both subgeneric and subspecific names are used) names are more cumbersome, than practical. An efficient naming strategy offering a practical alternative is to use infor-mal species groups, as implemented in the jewel beetles Agrilus Curtis, 1825 (the larger animal genus with over 3,000 valid species, Jendek and Grebennikov 2011), or as it prevails in arranging 1,665 species of Drosophila Fallén, 1823 fruit flies, the classical object of biological studies (O'Grady and Desalle 2018). Only in a few cases do subgeneric names in hyperdiverse genera appear justified, and only in situations when a stable phylogeny has been first achieved (e.g. the ground beetle genus Bembidion Latreille, 1802 with over 1,300 species and over 50 subgenera, Maddison 2012, Maddison and Maruyama 2019; the ground beetle genus Carabus Linnaeus, 1858 with 940 species and 91 subgenera, Deuve et al. 2012; the fruit fly genus Drosophila Fallén, 1823 with 1,646 species taxonomically arranged in a number of subgenera and informal species group, O'Grady and Desalle 2018). Considering the above, both non-nominotypical Typoderus subgeneric names are herein synonymised and, therefore, the subgeneric classification of the genus is scrapped. Available evidence. The genus Anchonidium was recently reduced to include only five West Palaearctic species (of them two from Portugal recently described, Germann 2020), while all but two Anchonidium from the Afrotropical Region were transferred to a re-defined Aparopionella Hustache, 1939 (Grebennikov 2018). Two Anchonidium species described by Hoffmann (1968) from the Republic of the Congo as type species of two monotypic subgenera of Anchonidium were noted as belonging to neither Anchonidium nor Aparopionella (Grebennikov 2018). A decision on the taxonomic status of these four names was long delayed by the unavailability of the type series. These historical specimens were borrowed in 2007 from Hungarian Natural History Museum (=HNHM, Budapest, Hungary) by Nicolas Maughan (Aix-Marseille University, Marseille, France; loan #4240/7343) and not returned as of September 2019, despite numerous demands (personal communication, Ottó Merkl, curator of HNHM Coleoptera collection). Requests for high-resolution images of the type specimens resulted in a low-resolution dorsal view of the holotype of A. distinctum (Fig. 1). The image revealed a weevil consistent with Typoderus in its appearance (parallel-sided body, effaced elytral shoulders, two longitudinal ridges on each side of pronotum, of them the internal one zig-zag shaped). The last feature is synapomorphic for the clade of Typoderus plus Lupangus. The beetle in Fig. 1 differs from Lupangus by having an anteriorly directed rostrum (more ventrally directed in Lupangus), by lacking transverse dorsal groove behind eyes (present in Lupangus) and by the more rounded eyes (vertical in Lupangus, fig. 4 in Grebennikov 2017). These considerations, together with sympatry and great similarity of this species with A. baloghi ("extrêmement voisin de baloghi...", Hoffmann 1968: 24) are the only data available to conclude that both species taxonomically belong to the genus Typoderus and are, therefore herein transferred to the later as Typoderus distinctus (Hoffmann, 1968) comb. nov. and Typoderus baloghi (Hoffmann, 1968) Table 4. Character matrix with discrete morphological characters for diagnostics of new Cameroonian Typoderus weevils (see also Discussion). -Characters: 1. -Body, colour of darkest specimens (Fig. 3, that is of a beetle at least a few days old, because specimens freshly emerged from the pupa are much paler): greyish: 0; brownish: 1; blackish: 2.  Table 4. Sister clade to T. canthus sp. nov. and T. tela mon sp. nov. (Fig. 3). Externally similar to sympatric T. canthus sp. nov.; can be distinguished by presence of additional protuberance on elytral interstria 2-3 located on elytral declivity nearest to elytral apices ( Fig. 5B versus Fig. 6B).  Table 4. Sister clade to T. telamon sp. nov. (Fig. 3).     Table 4. Sister clade to T. amphion sp. nov., T. telamon sp. nov. and T. clytius sp. nov. (Fig. 3).

Inter-and infra-specific variability
The morphological interspecific variability is reflected by the character states in table 4. Several characters also vary within species, for an example the colour of the beetles varies depending on the age of the specimens. A freshly emerged beetle is paler than the colour referred to in character 1 (table 4). Similarly, not all specimens have the additional apical projection near inner apical spur in the hind tibiae referred to in character 3, nevertheless it is coded as present if at least some males have it. Lastly, the similarity of the pilosity on the elytral declivity to that on the rest of the elytra referred to in character 4 cannot be assessed in abraded specimens. The observed molecular variability within the newly described Typoderus species is consistent with the somewhat arbitrarily 1-2% threshold (Hebert et al. 2003a), although T. iphitus shows a deep split into two BINs differing by as much as 2.9%, indicating the possibility that it might consist of two cryptic species.

Conclusions
This is the first report of Typoderus weevils from Cameroon, where unnamed congeners are likely to exist. Thus, species-level identification of these beetles might be far from straightforward. A field biologist must collect as many Typoderus specimens as possible, in order to asses two characters with intraspecific variation (Table 4, particularly the colour of specimens). For determination of recently hatched pale individuals, molecular data from as in this work might be necessary. This study highlights two taxonomic challenges or, rather, severe practical limitations of standard taxonomic procedures when performed in inadequately known and likely exceptionally diverse clades, such as Typoderus weevils. Firstly, for more than a decade the type specimens anchoring two genus-group names (the subgenera of Anchonidium herein synonymised with Typoderus) are not returned to a public institution, rendering both names vulnerable to misinterpretation. Secondly, even if perhaps not as diverse as the weevil genus Trigonopterus with well over a thousand unnamed species (Riedel et al. 2013), many dozens of unnamed Typoderus have emerged during extensive litter sampling in Tanzania (Grebennikov 2017(Grebennikov , 2019b. The effort to merely document this vast new diversity following the current taxonomic procedures is in itself an overwhelming task, for which no resources seem readily available. Being restricted by uncertainties surrounding old taxonomic names while facing a long list of new ones to introduce, I question when one shall find time and energy to utilize this yet-to-befine-tuned taxonomy as a tool for evolutionary biological studies? Perhaps similarly to astronomy, biology at present rapidly generates exponentially increasing amount of factual biodiversity data, for which the classical information storage system, the Linnaean taxonomy, is gradually becoming inadequate (comparably to the Messier or other historical catalogues of astronomic objects, presently of mainly educational interest). Will emerging DNA-based identifiers, such as BINs, take over species names? Only the future will tell.

Acknowledgements
Nicolas Maughan (Marseille, France) took and made available image of the holotype of Anchonidium distinctum shown in Fig. 1 (herein referred to as Typoderus distinctus comb. nov.). Václav Gvoždík (Prague, Czech Republic) shared his opinion on the timing of CVL animal speciation.
He and Adam Brunke (Ottawa, Canada) critically read earlier versions of the manuscript prior to its submission. I thank an anonymous reviewer and Michael Schmitt for helpful reviews. Anna Hundsdörfer (Dresden, Germany) edited the manuscript of this paper and made suggestions, many of them are thankfully incorporated.