Research Article |
Corresponding author: Milan Zúbrik ( zubrik@nlcsk.org ) Academic editor: John Midgley
© 2019 Milan Zúbrik, Sandrine Picq, Philippe Oremans, Audrey Nisole, Sophie Tremblay, Michel Cusson, Ľubomír Panigaj, Barbora Mikitová, Maurizio Bollino.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Zúbrik M, Picq S, Oremans P, Nisole A, Tremblay S, Cusson M, Panigaj Ľ, Mikitová B, Bollino M (2019) Morphological and genetic diversity of two individual forms of Euphaedra eberti (Lepidoptera, Nymphalidae). African Invertebrates 60(2): 195-213. https://doi.org/10.3897/AfrInvertebr.60.35262
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A total of 385 Euphaedra eberti Aurivillius, 1898, adults collected between 2012 and 2018 in the vicinity of Bangui, Central African Republic, were examined for intraspecific morphological variability, genetic diversity and genitalia structure. The species shows significant wing pattern variability. Two main morphotypes were identified in the set: the nominate form eberti, and the one comprising specimens with a red patch, form rubromaculata. However, both forms had similar genitalic structures and shared some specific wing marks, in addition to displaying the same COI (i.e., barcode region of the mitochondrial cytochrome c oxidase subunit I gene) haplotype, strongly suggesting that the two morphologically distinct forms belong to the same species, E. eberti. The causes of this variability remain unclear.
Limenitidinae, Euphaedra taxonomy, male and female genitalia, color variation, COI gene sequencing
The genus Euphaedra Hübner, 1819 (Lepidoptera, Nymphalidae), with about 200 recognized species, is one of the most speciose butterfly genera in Africa. The majority of species within this genus are true rain forest insects that live under the tree canopy. Despite the fact that these species form one of the most colorful, and one of the largest groups of butterflies on the African continent, many unresolved issues about their taxonomy persist (
Euphaedra eberti Aurivillius, 1898 (Lepidoptera, Nymphalidae) belongs to the group themis (
Two subspecies of E. eberti have so far been described.
In 1920, Schultze described an E. eberti colorful form based on a single female collected by himself in Duna (Ubangi, Belg. Kongo, presently DRC), and with a different color pattern by comparison to the species described by
The two above individual forms, i.e. the nominate form E. eberti eberti, without the red patch, and the red-wing form, E. eberti ab. rubromaculata, display large differences in their wing patterns. Therefore the question may be raised as to whether they should be considered two separate species, perhaps wrongly described as a single species by earlier authors (
The study material – adults of E. eberti – were provided by a local collector. They were collected using entomological nets and fruit-baited traps between 2012 and 2018, in an area west of the Ubangi River, south west from Bangui, in CAR. Dead adult specimens were shipped to the laboratory of Forest Research Institute Zvolen (Slovakia). The specimens were set and subsequently identified based on external morphological characters, using relevant identification keys (
Butterflies were collected at 11 locations: Bangui: 4°22'24.0"N; 18°35'15.6"E, Maka: 4°16'58.9"N; 18°28'42.4"E, Mbata: 3°41'28.4"N; 18°18'21.7"E, Mbaiki: 3°50'32.7"N; 17°59'37.0"E, Moloukou: 3°44'15.2"N; 17°32'09.3"E, Bimon: 4°19'41.4"N; 18°18'52.1"E, Boukoko: 3°53'44.7"N; 17°54'30.7"E, Pissa: 4°03'36.1"N; 18°12'42.6"E, Bokassi: 4°12'56.3"N; 18°34'59.8"E, Bouchia: 3°45'41.1"N; 18°10'48.4"E and Mokpoto: 4°15'33.3"N; 18°31'54.1"E.
Male and female genitalia were dissected using standard dissection protocols (
For genetic analyses, we considered 32 butterflies (16 females and 16 males) of the nominate form and 32 butterflies (16 females and 16 males) of the ab. rubromaculata. DNA was extracted from one leg taken from each mounted butterfly, using the DNeasy Blood & Tissue Kit (Qiagen, Carlsbad, CA, USA), according to the manufacturer’s instructions. DNA quality was assessed using a Nanodrop ND-1000 spectrophotometer (ThermoFischer, Waltham, MA, USA).
The barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified for each butterfly using two primers: “LCO” (forward primer; 5’-GGTCAAATCATAAAGATATTGG-3’) and “HCO” (reverse primer; 5’- TAAACTTCAGGGTGACCAAAAAATCA-3’) (
Sequences were aligned, read and corrected using the GeneStudio software (GeneStudio inc. 2011, http://genestudio.com/). For each individual form, we used the Mega7 software (
In addition, we constructed a maximum likelihood (ML) phylogenetic tree, using the General time reversible (GTR) model. In constructing this tree, we included four Euphaedra outgroup species: E. zaddachi Dewitz, 1879, E. herberti Sharpe, 1891, E. eusemoides Grose-Smith & Kirby, 1889 and E. uganda Aurivillius, 1895. These species were selected on the basis of COI sequence availability in GenBank (E. zaddachi, KU219620.1; E. herberti, AY218241.1; E. eusemoides, KU219618.1, E. hewitsoni, MG741027.1, E. spatiosa, MG741121.1 and E. uganda, KU219619.1). Finally, we conducted an analysis of molecular variance (AMOVA) to statistically assess whether there existed a genetic difference between the red and nominate form.
Finally, median joining networks (
A total of 385 specimens of E. eberti were analyzed during this study, and two significantly different individual forms were identified: the nominate form eberti (Figures
E. eberti eberti, adults, males. 1 recto, 7 verso (Bangui; 2014); 2 recto, 8 verso (Bauchia; 14 Dec. 2016); 3 verso, 9 recto (Maka; 11. Mar. 2017); 4 recto, 10 verso (Bangui; 4 Mar. 2016); 5 recto, 11 verso (Boukoko; 20 Jun. 2015); 6 recto, 12 verso (Bimon; 8 May. 2017). Arrows point to structures referred to in the results section.
Wingspan: 6.0–7.2 cm (n = 16, x‒ = 6.51 cm, SD = 0.31). The dorsal side appears metallic green (Figure
Male genitalia (Figure
Wingspan: 7.6–9.0 (n = 16, x‒ = 8.19 cm, SD = 0.33). FWD dorsal ground color black with a gray-blue-green (Figure
Female genitalia (Figure
Wingspan: 5.0–7.2 cm (n = 16, x‒ = 6.43 cm, SD = 0.45, not significantly different from that of E. eberti eberti males). Metallic green on FWD (Figure
E. eberti ab. rubromaculata, adults, males. 1 recto, 7 verso (Mokpoto, 22 Jul. 2017); 2 recto, 8 verso (Mokpoto; 22 Jul. 2017); 3 verso, 9 recto (Bangui; 2016); 4 recto, 10 verso (Bangui; 2014); 5 recto, 11 verso (Boukoko; 9 Jun. 2018); 6 recto, 12 verso (Bokassi; 10 Jun. 2016). Arrows point to structures referred to in the results section.
Male genitalia (Figure
Wingspan: 7.7–9.2 cm (n = 16, x‒ = 8.42 cm, SD = 0.40; not significantly different from that of E. eberti eberti females). FWD dorsal ground color black with a yellow-green (Figure
Female genitalia (Figure
We successfully obtained the COI barcode sequence (572 nucleotides) for 47 specimens (25 red and 22 nominate). See Table
The genetic diversity indices computed here (PVS, π, genetic distance) were similar for these two individual forms (Table
Individual form | n | Female | Male | Variable site | PVS | π | Genetic distance* |
---|---|---|---|---|---|---|---|
rubromaculata | 25 | 14 | 11 | 12 | 0.02083 | 0.008484 | 0.009 |
eberti | 22 | 11 | 11 | 15 | 0.02604 | 0.007456 | 0.008 |
Total | 47 | 25 | 22 | 19 | 0.03299 | 0.008387 | 0.008 |
The phylogenetic tree. COI-based ML tree constructed using the GTR model. Red dots indicate rubromaculata form (the others are nominate form). The six outgroups are E. zaddachi (KU219620.1), E. herberti (AY218241.1), E. eusemoides (KU219618.1), E. uganda (KU219619.1), E. hewitsoni (MG741027.1) and E. spatiosa (MG741121.1).
Median-joining networks constructed for the 15 E. eberti COI haplotypes obtained in the present study, as a function of A sampling location B wing color form, and C sex. Each colored circle represents a specific haplotype identified by the number beside it, and the size of each circle is proportional to the number of sequences for that haplotype. The network comprises two clusters separated by ~8 substitutions (cross-lines). Each network was generated using the software POPART.
Adults of E. eberti eberti are similar to those of E. preussi Staudinger, 1891, E. fascinata Hecq, 1984 and E. vicina Hecq, 1984, common in the investigated area and showing large color variations. However, using the broken and “S”-shape subapical band and some other morphological features, distinguishing E. eberti from these species proved easy. Euphaedra species that resemble E. eberti ab. rubromaculata, such as E. themis Hübner, 1807 or E. permixtum Butler, 1873, do not occur in the investigated area. Among the species found in the investigated area, the one most similar to ab. rubromaculata is the rare E. campaspe Felder & Felder, 1867, which lacks the large red patch (occurring only in extremely rare cases and never with the intensity observed in E. eberti ab. rubromaculata) on the FWD, therefore making misidentification unlikely.
Among rubromaculata individuals, we found several exemplars with blue color on the FWD and blue-yellow color of the HWD, among the more frequently observed yellow-green specimens. We have here confirmed that the ab. rubromaculata is present in both males and females, although no male representative of the blue form was found.
Observed dissimilarities in wing patterns between the two individual forms were not accompanied by significant differences in the genitalia, wingspan or COI haplotypes. This strongly suggests that the two individual forms reported here represent a natural variation within populations of a single species, thereby supporting current Euphaedra taxonomy (
Because the collection date was not recorded for every specimen, we could not evaluate the possibility that the two forms described here could be the outcome of seasonal polymorphism (polyphenism). Although we know the E. eberti ab. rubromaculata individuals from our collection were most prevalent in June and August (data not shown), pointing to possible seasonal fluctuations, our data are too sparse to draw conclusions at this stage. The only other report on seasonal occurrence is
Beyond seasonal polymorphism, other types of intraspecific wing pattern polymorphism have been reported for the Lepidoptera (
The two individual forms, i.e. the nominate form E. eberti eberti, without the red patch, and the red-wing form, E. eberti ab. rubromaculata, display large differences in their wing patterns, which initially suggested they should be considered separate species. However, both forms had similar genitalic structures and shared some specific wing marks, in addition to displaying the same COI haplotype, strongly suggesting that the two morphologically distinct forms belong to the same species, E. eberti. The results of our investigation are in agreement with the current taxonomy of the genus Euphaedra. The causes of the observed variability remain unclear.
The authors thank Bangake Gilbert (RCA) for providing the study material, Reza Zahiri for technical assistance with the POPART software and Dominique Fournier (Canada) for linguistic and editorial improvements.