Corresponding author: Łukasz Przybyłowicz (
The subgenus
Paśnik A, Tarcz S, Przybyłowicz Ł (2023) A review of the subgenus
For some time now, there has been an increasing number of in-depth studies of African
Besides purely α-taxonomy publications, more comprehensive revisionary studies of species groups and genera have been published recently, including those devoted to tiger moths (
A unique example of a large scale ecological project is the one focused on the study of Mount Cameroon, which is an active volcano, on southwestern flanks covered with primary tropical rainforest. It represents one of the well-known biodiversity hotspots, with an exceptionally high number of recorded taxa (
One of the groups of moths from this region deserving to be studied in detail is genus
Currently, three subgenera are recognized within the genus:
The biology, including the food plant(s) of
A fairly large problem is the abovementioned morphological uniformity across the genus, resulting in the small number of well-defined distinguishing characters that may be used to separate the species. Thus, a reliable determination is only possible with large series and access to reference specimens, best with the combination of morphological and molecular methods. This relatively new approach, known as integrative taxonomy, is based on the idea that results of different methods should be combined to strengthen the taxonomic hypotheses (
The aim of this paper is to revise the subgenus
Analysed specimens of the genus
Each specimen was photographed using a Canon 70D digital camera with a macro lens EF 50 mm. Genital slides were made from 70 individuals (64 males and 6 females). Abdomens were detached from selected specimens and macerated in 10% KOH solution in a water bath for about 30 minutes. Next, each abdomen was transferred to a petri dish with distilled water and a drop of liquid reducing surface tension. Scales were removed from the abdomen with a fine and thin brush. The cleaned abdomen was transferred to a new petri dish and unnecessary soft tissues were removed with entomological pins. Soft membranes, i.e. parts of aedeagus and female preparations were stained with chlorazol black. If possible, vesica was everted from the aedeagus. After the preparations were made, the specimens were labeled and the preparations were stored on basal slides in glycerin. When the comparative analyses are completed, the slides will be permanently encapsulated in Euparal (Agar Scientific, Essex, UK) and included in the collection. Pictures of the slides were taken using a stereoscopic microscope Leica S9i system. Images were adjusted with the Adobe Photoshop CC program. The morphology terminology follows
For wing scales examination one specimen was selected from each species:
From the surface of the wings, the scales were scraped into a dish with alcohol using a moistened entomological pin. Permanent slide preparations were made using Marc André II (
The differences in scales have been visualized by use of the scanning electron microscope. The specimens were selected, one specimen from each of the three species. Using a binocular microscope, scales were gently scraped from the wing fragments with an entomological pin. From the scales, three types of preparations were made, one on a basic slide where the material was embedded in glycerin. Mounted microscope slides were made using Marc Andre II mounting medium. A separate preparation has been made for SEM images. The scales were glued onto carbon glue holders and covered with gold using an Ion Sprayer JEOL JFC-1100E. For taking photos a scanning microscope JEOL JSM5410 with tungsten cathode was used. The images were taken at the Institute of Geological Sciences at the Jagiellonian University, Kraków, Poland.
Specimens collected not earlier than about 10 years ago were selected for DNA isolation. From each dried specimen one or two legs were sampled. The isolation of DNA was done with the NucleoSpin Tissue kit (Machery-Nagel, Germany), following the manufacturer’s protocol.
Sequence of the barcode part of the mitochondrial gene cytochrome c oxidase subunit I (
The ready hot-start PCR mix (StartWARM HS-PCR Mix, A&A Biotechnology, Poland) was used. PCR reactions were performed in a total volume of 10 μl. The amplified products were electrophoresed in 1% TBE agarose gel for 30 min at 100 V and visualized under UV. PCR products were purified with Exo-BAP mix (EURx, Poland), following the standard protocol. Then successful PCR products were sequenced in both directions using the same primers as for PCR reaction (LEP-F1/LEP-R1). For sequencing BrilliantDye v3.1 Terminator Cycle Sequencing Kit (NimaGen, the Netherlands) was used. Sequence reading was done with the use of an ABI Prism 3130xl sequencing machine in the Laboratory of Molecular Techniques at
For old, historic specimens of
For these samples primers LEP-F1/LEP-R1 failed, and additional PCR reaction was carried out using primers ZBJ-ArtF1c (5’-AGA TAT TGG AAC WTT ATA TTT TAT TTT TGG-3’) and ZBJ-ArtR2c (5’-WAC TAA TCA ATT WCC AAA TCC TCC-3’) for a short fragment of the
DNA sequences generated during this study are deposited in the GenBank database, and the accession numbers are provided in Table S1, Table S2.
The p-distance (Table S3) between barcode sequences was calculated in MEGA11 (
The
The
The discrete gamma distribution of rates among sites was applied (
All obtained trees were visualized with FigTree 1.4.3 (
The analysis of haplotype diversity (
Phylogenetic tree showing the variation of
Phylogenetic tree based on Bayesian interference method including
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Forewing silver-grey, opaque, never semi-transparent (Fig. |
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– | Forewing greyish-ochraceous, always at least semi-transparent, matt, or indistinctly shiny (Fig. |
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Forewing semi-transparent (Fig. |
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– | Forewing almost transparent (Figs |
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Process of valva elongate, narrow; vesica with single, well developed cornutus (Fig. |
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– | Process of valva invisible or in form of minute tubercle, vesica without cornuti |
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Uncus constricted before terminal bifurcation; terminal lobes shorter than one quarter of the length of uncus (Fig. |
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– | Uncus with parallel margins not constricted before terminal bifurcation; terminal lobes approximately the half the length of uncus (Figs |
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Distal, submedial sclerotization of VII sternite Y-shape, significantly longer than wide (Fig. |
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– | Distal, submedial sclerotization of VII sternite Y-shape, approximately as long as wide (Fig. |
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Signum heavily sclerotized, irregular, suboval, at most twice as long as wide (Fig. |
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– | Signum sclerotized, irregular, elongate, at least twice as long as wide (Fig. |
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The subgenus differs from the two remaining subgenera viz.
Subgenus
Haplotype network of
Taking into account lack of well-defined pattern on wings and body which might serve as a source of diagnostic characters, the differentiations between darker and paler coloration used in descriptions should be treated with reservation. The degree of colour saturation varies significantly depending on illumination, which combined with the overall uniformity of specimen coloration makes it very difficult to objectively compare darker and paler structures and the intensity of the differences.
Overview of the genital apparatus of males
Compilation of female genital organs
Details of female genitalia with both a marked antrum and a sclerotization of VII sternite. The arrow marks the location and sclerotization of the sternite. From left
Due to the extreme similarity of all members of
Differences in the scale morphology of wing region between CuA1 and CuA2 near DC of
Male genitalia allow for an easy separation of
Female genitalia examined are partly damaged and incomplete. Additionally, they are unknown for several other
Head. Frons and vertex pale ochraceous; labial palpus darker, three segmented of which the second is the longest and the last directed downwards, densely covered with narrow scales; scapus pale ochraceous; flagellum bipectinate, concolorous with scapus; flagellomeres honey; eye convex, indistinctly ovoid. —
(Fig.
(Figs
Difficult to assess. Three males come from the same sampling (place, time), the fourth one is much older, slightly damaged and faded. Within the three males from Wack, only some very indistinct variation in the intensity of the ochraceous coloration of the wings and body can be detected. Genitalia differ in the shape of elongate saccus which may have parallel or slightly concave lateral margins and rounded or triangular termination.
Weakly expressed and in available material reliably visible only in the length of rami of antenna which in female are approximately three times shorter than in male counted at central portion of antenna. Male from Adamaua has M2–M3 of forewing on a short stalk.
(Fig.
Distribution of
The specific epithet “
(84 ♂♂, 4 ♀♀) ♂ West Africa, Liberia, Gbarpolu County, Gola National Forest, Kungbor, Nordrand, 401m,
Forewing length 12 mm. —
(Figs
Overview of variation in the bifid termination of uncus in
(Figs
Very variable species in the intensity of dark tint of the body. The holotype male represents the pale, almost ‘whitish’ colour morph while some specimens can be much darker up to almost completely ochraceous with all intermediate forms. The darker, indistinct pattern can be also observed in the pale specimens especially on different portions of head as frons or vertex. The male genitalia also express significant variation in morphology of uncus and especially the development and perspicuity of its forked termination. The detailed examination of detected variability is elaborated in the discussion part of the article.
The female differs from the male by much shorter rami of antenna which are twice as long as the width of the antenna and by shorter epiphysis reaching at most 2/3 of the foretibia length.
(Fig.
The details of the holotype locality are extracted from the label and were not published together with the original description.
Holotype: ♀ “Lake Tshohoa, Ruanda Dist., Cent. Afr. Aug. ‘19, T.A. Barns; Joicey Bequest. Brit. Mus. 1934–120”; g.s. ARCT 5795 [
♂
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Vestiture unicolorous pale ochraceous expressing darker or lighter tint depending on the illumination; legs of the same uniform coloration; epiphysis stout reaching 4/5 of the foretibia length; mid and hind tibia with a pair of short, terminal spurs. —
Entirely pale ochraceous, concolorous with the rest of the body. Upperside similar to underside. —
Opaque, densely covered by flattened scales with distinct, clearly visible shine on the entire surface of the wing; scales suboval, moderately elongate, with rounded terminal margin, pale ochraceous, slightly darker along veins, with admixture of white-creamy ones in areas between them; subcostal zone up to DC indistinctly darker than remaining part of wing; veins covered by scales; cilia pale cream; R1 separated from R2–R5; M2–M3 narrowly separated; distances between M3- CuA1 and CuA1–CuA2 similar; 1A+2A almost straight, without distinct curve in one third of its length; coloration of underside similar to upperside, retinaculum present. —
Coloration somewhat paler than in forewing; cilia almost white, Rs–M1 completely fused.
(Fig.
(Figs
The limited number of specimens does not allow for a proper detection of individual variation. Among the examined females it is expressed by differences in forewing length and intensity of ochraceous coloration, which may be more or less pale. Additionally, in some specimens, both males and females, the fused Rs–M1 can be forked before the termination.
The female differs from the male by much shorter rami of antenna which are twice as long as the width of antenna and by shorter epiphysis reaching at most 2/3 of the foretibia length.
(Fig.
The detailed description of
The taxonomic interpretation of 89 specimens of
The remaining two species are represented in our study in too few specimens to observe any clear morphological variability, except the most common referring to the indistinct differences in wing length and intensity of coloration. Much larger sets of specimens are necessary to investigate this aspect, however it is unlikely that any of the two taxa is as polymorphic as is
To carry the phylogenetic analyses two methods were used –
The
To assess the intraspecific genetic variability of
The number of specimens available for this genetic study was limited to three specimens collected in the same locality. The p-distance between them varies between 0.0 and 0.9%.
The interspecific p-distance between
The interspecific distance between
Haplotypes were obtained for 51 sequences of
The concept of dividing
The study and revision of the remaining taxa of the genus
Our study of specimens of
The DNA barcodes obtained from museum specimens are very useful to resolve taxonomic uncertainty with the type species and some cryptic species, especially if morphological data on its own is insufficient (Hernandez-Triana 2014). Often the species of interest had been collected long before the DNA sequencing started to be a frequently used technique in biodiversity studies and describing new species (
The range of the species
The most well-known examples of polymorphism are: sexual dimorphism, polyphenism, color polymorphism, and geographic polymorphism (
Also, we cannot rule out a hypothesis that the observed polymorphism is a result of existence of several (at least two) closely related cryptic species. However, getting a clearer picture favouring or falsifying this hypothesis would require access to a much larger set of specimens, covering more or less evenly the entire range of the taxon in question. Finally, the observed variability may be a result of an ongoing diversification process, although this hypothesis is unlikely given the fact that none of the forms is geographically restricted or can be linked with any environmental factor like altitude or type of vegetation.
It can be also assumed that the high morphological variability is maintained within populations as the response for the wide distribution and the utilization of the very different microhabitats on the large area stretching from almost sub-Saharan western Africa, through the costal equatorial areas along the Guinea Bay up to the again semiarid uplands of Angola.
Scales and their structures are one of the most studied photonic structures for a long time (Mouchet et al. 2018). Detailed study of the
SEM image showing the differences between scale shape of
Differences in the forewing transparency.
The range of the subgenus
The location and biome of the Cameroon’s mountains, including Adamawa, make it one of West Africa’s biggest hot spots (
Contrary to the abovementioned species
To sum up the biogeographic aspects, it should be noted that only members of the subgenus
We revised the subgenus
The new species unexpectedly appeared to be unique among all other known
Finally we would like to stress that
We are indebted to Gyula László (African Natural Research Trust), Michael Ochse, Sáfián Szabolcs, Thomas Witt, Knud Larsen for providing access to
Table S1
Table S2
Table S3