Research Article |
Corresponding author: Yoshitaka Kamimura ( kamimura@keio.jp ) Academic editor: Fabian Haas
© 2023 Yoshitaka Kamimura, Chow-Yang Lee, Junsuke Yamasako, Masaru Nishikawa.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kamimura Y, Lee C-Y, Yamasako J, Nishikawa M (2023) Identification and reproductive isolation of Euborellia species (Insecta, Dermaptera, Anisolabididae) from East and Southeast Asia. ZooKeys 1146: 115-134. https://doi.org/10.3897/zookeys.1146.98248
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Euborellia (Anisolabididae: Anisolabidinae) is one of the most speciose genera of earwigs (Dermaptera), and its species-level classification is difficult. To settle the classification of brachypterous species with abbreviated tegmina recorded from East and Southeast Asia, we examined the morphology and reproductive isolation of three tentative Euborellia species, and analyzed the DNA barcoding region of the mitochondrial cytochrome oxidase subunit I (COI) gene. The observed complete reproductive isolation among the three Euborellia taxa and considerable differentiation in the COI sequences clearly show that each should be treated as a separate species. Based on morphology, distribution and the DNA sequence, we identify Euborellia sp. 1 of Malaysia as E. annulata (Fabricius), a circumtropical cosmopolitan with no records of a fully winged form. Samples from Ioto Island (= Iwo-jima Island: Ogasawara Islands, southern Japan) were also identified as this species. Euborellia sp. 3, from the main islands of Japan, was generally larger and lacked a Y-shaped pigmented area on the penis lobe, which is characteristic of Euborellia sp. 1. We propose reinstating E. pallipes (Shiraki) as the oldest name for this taxon. Euborellia sp. 2, even the brachypterous form, can be distinguished from these two species by its paler coloration (particularly the femora), ecarinate post-abdomen, and the shape of the male genitalia (parameres). We tentatively identify this species as E. philippinensis Srivastava based on the morphology of the brachypterous form, although the macropterous form cannot be distinguished from E. femoralis (Dohrn).
DNA barcoding, Euborellia pallipes, genital morphology, post-copulatory reproductive isolation, reinstatement
Euborellia Burr, 1910 (Anisolabididae: Anisolabidinae) is one of the most speciose genera of earwigs. It includes approximately 50 species (
This study examined the relationships among three tentative Euborellia species with flap-like vestigial tegmina (= forewings) found in East and Southeast Asia (named Euborellia sp. 1, Euborellia sp. 2 and Euborellia sp. 3 in the rest of the article). Euborellia sp. 1 was recorded from Malaysia, and was tentatively identified as Euborellia annulata (Fabricius, 1793) in
An apterous species of Euborellia was recently discovered as a possible intruder in Europe (
Two experiments examined pre- and post-copulatory reproductive isolation among the three tentative species. Virgin females were obtained by separating newly emerged adults every three days from laboratory cultures of nymphs (wild-caught or mainly the F1 generation). For Euborellia sp. 1 and Euborellia sp. 3, individuals derived from five localities of Malaysia (Batu Ferringi [5.47°N, 100.25°E], Bukit Bendera [5.42°N, 100.26°E], Bayan Indah beach [5.34°N, 100.31°E], and Bayan Lepas [5.33°N, 100.31°E] of Penang Island and Kuantan [3.80°N, 103.34°E], Pahang state: and some of their hybrid F1) and three localities from Japan (Tokushima city, Tokushima Prefecture [34.12°N, 134.58°E], Yokohama city, Kanagawa Prefecture [35.51°N, 139.57°E], and Komae city, Tokyo Prefecture [35.63°N, 139.57°E]) were used, respectively. All samples of Euborellia sp. 2 were derived from a single locality (Sungai Nipah, Penang Island, Malaysia [5.32°N, 100.20°E]), but pairing of a male and a female from the same full-sib family was avoided. For males, wild-caught adults were also used (see Suppl. material
In the first experiment (Exp. 1), a virgin female (age: 5–68 days after imaginal eclosion: median = 9 days) was paired with a conspecific or heterospecific male in a plastic container (50 × 32 mm, 12 mm high) with plaster of Paris at the base for 21 h (N = 5 for each species combination). Then the females were sacrificed by placing them in a freezer (−20 °C) for later examination of their insemination status. The spermatheca was dissected from the females in insect Ringer’s solution (0.9 g NaCl, 0.02 g CaCl2, 0.02 g KCl, and 0.02 g NaHCO3 in 100 mL water) under a stereomicroscope (EZ vision, Saxon, Guangzhou, China), and then examined under a light microscope (BX53 or CX21, Olympus, Tokyo; 40–400×). In the second experiment (Exp. 2), a virgin female (age: 3–83 days after imaginal eclosion: median = 6 days) was paired with a conspecific or heterospecific male in a separate plastic vessel (60 mm diameter, 40 mm high) for 72 h (N = 5 for each species combination). Then the females were reared separately in the vessel for 30 days after removing the male. Oviposition and hatching of offspring were checked every two or three days. The spermatheca of the females that produced no hatchlings during the observation period was examined for the presence of sperm, as described above. Females with at least one hatchling or sperm in the spermatheca were scored as “inseminated”.
The external morphologies of dried adult materials were examined under a stereomicroscope (S8-APO; Wetzlar, Germany or SZX16; Olympus, Tokyo, Japan) and photographed using an Olympus Pen e-pl1s digital camera (Olympus). “Microscope mode” and “Focus-stacking sub-mode” of a Tough-TG5 digital camera (Olympus) were also used to obtain composite images of the external traits. The male genitalia were extracted from freeze-preserved, dried, or fresh specimens anesthetized with carbon dioxide under a stereomicroscope. After mounting on a glass slide with insect Ringer’s solution, they were observed and photographed under a light microscope (BX53, 100–400×; Olympus) equipped with an Olympus DP80 CCD camera or a differential interference contrast (DIC) microscope (BX53, 100–400×; Olympus) fitted with an Olympus Pen e-pl1s digital camera. Based on photographs taken under the DIC microscope, selected parts of each image in focus were composed using Combine ZP Image Stacking Software (
The samples were wild-caught from Penang Island (Bayan Lepas [Penang-1], Batu Ferringi [Penang-2], and Bayan Indah beach [Penang-3]) for Euborellia sp. 1. For Euborellia sp. 2 and Euborellia sp. 3, samples of laboratory stock populations, derived from a female collected from Sungai Nipah, Penang Island, Malaysia (in 2012), and Takasago city, Hyogo Prefecture, Japan [34.75°N, 134.80°E] (in 2018), respectively, were examined. For Euborellia sp. 3, wild-caught and mainly F1-generation offspring were also examined for the following seven localities of Japan: Satsuma-sendai city, Kagoshima Prefecture [31.81°N, 130.31°E: Kagoshima-1], Shimokoshiki Island, Kagoshima Prefecture [31.66°N, 129.72°E: Kagoshima-2], Naruto city, Tokushima Prefecture [34.20°N, 134.60°E], Shizuoka city, Shizuoka Prefecture [35.01°N, 138.39°E: Shizuoka-1], Izunokuni city, Shizuoka Prefecture [35.06°N, 138.95°E: Shizuoka-2], Yokohama city, Kanagawa Prefecture [35.51°N, 139.57°E], and Iwaki city, Fukusima Prefecture [36.88°N, 140.79°E].
For Euborellia sp. 1 and 3, which were challenging to discriminate based on their external appearance, three traits were chosen for measurement based on the results of a pilot study: maximum head width (including eyes), maximum pronotum width, and hind tibia length. These traits can usually be measured on dried specimens preserved in museums and can be used for future studies on this group. These traits were measured for dried materials (Suppl. material
In addition to the samples collected by the authors, two female and one male adult Euborellia collected from Ioto Island (= Iwo-jima Island) in the Ogasawara Islands (= Bonin Islands) preserved in the collection of
Kanagawa Prefectural Museum of Natural History (
Total genomic DNA was extracted from fresh, ethanol-preserved, or dried samples of Euborellia and other dermapterans (Suppl. material
Multiple sequence alignments were conducted with ClustalW (
The crossing experiments revealed that the three tentative Euborellia species are strongly isolated (Fig.
Insemination (a, b), oviposition (c), and hatching success (d) of conspecific and reciprocal heterospecific crosses among the three tentative species of Euborellia: Euborellia sp. 1 (1), Euborellia sp. 2 (2), and Euborellia sp. 3 (3). These species are identified as E. annulata, E. philippinensis, and E. pallipes, respectively.
The external morphologies of the three tentative species, particularly those of Euborellia sp. 1 and 3, are similar and difficult to distinguish. Except for the fully winged morph of Euborellia sp. 2 (Fig.
Female habitus (a–c), head and thorax of a fully winged-form male (d), base of female right antenna (e–g), female right hindleg (h–j), male forceps (k–m), left side of male post-abdomen (n–p), and distal part of male genitalia (q–s) of Euborellia sp. 1 (identified as E. annulata; a, e, h, k, n, q), Euborellia sp. 2 (identified as E. philippinensis; b, d, f, i, l, o, r), and Euborellia sp. 3 (identified as E. pallipes; c, g, j, m, p, s). Femoral and tibial black marking are indicated by the red and orange arrows, respectively (h–j). Carination and dorso-lateral angles of the abdominal tergites, the latter forms the lateral ridges, are indicated by the yellow and light blue arrowheads, respectively (n–p). On each penis lobe, a pair of denticulated pads (the orange and magenta arrowheads) and a Y-shaped area of pigmentation (only in Euborellia sp. 1: black arrowheads) are present. The external apical angle of the parameres (pm) is acute in Euborellia sp. 2 (the black arrow). Scale bars: 5 mm (a–c); 1 mm (d–p); 500 µm (q–s).
The genital morphologies are also quite similar among the Euborellia species examined here. The male genitalia are elongated, almost the body length (Euborellia sp. 1 and 3) or the abdominal length (Euborellia sp. 2). On each penis lobe including a thin virga, two humps of denticulated pads are present (orange and magenta arrowheads in Fig.
To separate Euborellia sp. 1 and 3, three morphological traits, considered measurable in dried specimens from museums, were quantified and compared: the maximum head width, maximum prothorax width, and hind tibia length. Although the sample size is small for Euborellia sp. 1, the three traits were generally smaller in Euborellia sp. 1 than in Euborellia sp. 3, particularly in females (Fig.
Relationship between the maximum pronotum width and maximum head width (a, b), and relationship between the maximum pronotum width and hind tibia length (c, d) of female (a, c) and male (b, d) Euborellia species (Euborellia sp. 1 and Euborellia sp. 3). Red, blue, and black symbols represent samples from Malaysia, the main islands of Japan (Honshu, Shikoku, and Kyushu, and Shimokoshiki Island near Kyushu), or Ioto Island, respectively. The data of a female E. annulata, collected from Basse-Terre Island, Guadeloupe Archipelago, is also shown in a and c (light blue crossed diamonds). Details of the localities are provided in Suppl. material
As additional materials from Japan, two female and one male adult Euborellia collected from Ioto Island (= Iwo-jima Island) in the Ogasawara Islands (= Bonin Islands) were examined. The tegmina of these samples are small flaps (Fig.
Euborellia specimens collected from Ioto Island, Ogasawara Islands, Japan (a–f) and the type (holotype) material of Anisolabis pallipes Shiraki, 1905, preserved in Insect Museum, National Taiwan University, Taipei, Taiwan (g). a adult female b adult male c left lateral view of the male post-abdomen d right hindleg of the male e left hindleg of the female f part of the male genitalia (right paramere [pm] and the penis lobe). For the meanings of the arrows and arrowheads, see the caption of Fig.
Based on a brachypterous adult female collected from Takasago, Hyogo Prefecture, Japan,
Although comparable numbers of transversions (Tv) and transitions (Ts) are estimated to occur in the 2nd and 3rd codon positions at the genetic distance larger than ca 0.2 (Fig.
The percent sequence divergence was lower than 2% within each tentative Euborellia species, except for one individual of Euborellia sp. 1, which showed about 5% divergence from the other three conspecific samples (Table
Percent divergence (p-distance) between the sequences. Intraspecific comparisons are highlighted in a different color for each species.
Species (GenBank Accession No.) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | Euborellia sp. 1 (LC715987)* | 0.000 | ||||||||||||||||
2 | Euborellia sp. 1 (LC715988)* | 0.055 | 0.000 | |||||||||||||||
3 | Euborellia sp. 1 (LC715989)* | 0.052 | 0.015 | 0.000 | ||||||||||||||
4 | Euborellia sp. 1 (LC715990)* | 0.048 | 0.012 | 0.006 | 0.000 | |||||||||||||
5 | Euborellia annulata (LC740580) | 0.018 | 0.065 | 0.062 | 0.059 | 0.000 | ||||||||||||
6 | Euborellia sp. 2 (LC715959)** | 0.141 | 0.139 | 0.135 | 0.135 | 0.142 | 0.000 | |||||||||||
7 | Euborellia sp. 2 (LC715979)** | 0.141 | 0.139 | 0.135 | 0.135 | 0.142 | 0.000 | 0.000 | ||||||||||
8 | Euborellia plebeja (MW703670.1) | 0.153 | 0.155 | 0.152 | 0.150 | 0.146 | 0.097 | 0.097 | 0.000 | |||||||||
9 | Euborellia sp. (winged species:MW703671.1) | 0.144 | 0.151 | 0.150 | 0.150 | 0.142 | 0.109 | 0.109 | 0.087 | 0.000 | ||||||||
10 | Euborellia sp. 3 (LC715955)*** | 0.165 | 0.174 | 0.167 | 0.171 | 0.173 | 0.136 | 0.136 | 0.160 | 0.153 | 0.000 | |||||||
11 | Euborellia sp. 3 (LC715956)*** | 0.165 | 0.174 | 0.167 | 0.171 | 0.173 | 0.136 | 0.136 | 0.160 | 0.153 | 0.000 | 0.000 | ||||||
12 | Euborellia sp. 3 (LC715957)*** | 0.165 | 0.174 | 0.167 | 0.171 | 0.173 | 0.136 | 0.136 | 0.160 | 0.153 | 0.000 | 0.000 | 0.000 | |||||
13 | Euborellia sp. 3 (LC715958)*** | 0.168 | 0.177 | 0.170 | 0.174 | 0.173 | 0.139 | 0.139 | 0.158 | 0.154 | 0.006 | 0.006 | 0.006 | 0.000 | ||||
14 | Euborellia femoralis (MW703672.1) | 0.159 | 0.152 | 0.150 | 0.149 | 0.155 | 0.126 | 0.126 | 0.150 | 0.145 | 0.114 | 0.114 | 0.114 | 0.114 | 0.000 | |||
15 | Euborellia femoralis (MW703673.1) | 0.165 | 0.155 | 0.152 | 0.151 | 0.160 | 0.124 | 0.124 | 0.149 | 0.147 | 0.111 | 0.111 | 0.111 | 0.111 | 0.005 | 0.000 | ||
16 | Euborellia annulipes (LC731318) | 0.173 | 0.174 | 0.169 | 0.171 | 0.171 | 0.156 | 0.156 | 0.152 | 0.153 | 0.182 | 0.182 | 0.182 | 0.178 | 0.166 | 0.164 | 0.000 | |
17 | Euborellia arcanum (KP019208.2) | 0.171 | 0.174 | 0.168 | 0.168 | 0.168 | 0.142 | 0.142 | 0.167 | 0.161 | 0.188 | 0.188 | 0.188 | 0.192 | 0.165 | 0.166 | 0.179 | 0.000 |
Although the support is low (56%), the samples of Anisolabidinae (Anisolabididae) formed a monophyletic clade (Fig.
The maximum likelihood phylogenetic tree constructed from COI sequence data. Numbers at the branches indicate bootstrap values (% in 1000 replicates). GenBank accession numbers follow the localities in the parentheses. Euborellia sp. 1, Euborellia sp. 2, and Euborellia sp. 3 are shaded in red, yellow, and blue, respectively, indicating the proposed names. The details of the samples sequenced in the present study (the accession numbers beginning with “LC”), are available in Suppl. material
In the Euborellia clade, multiple samples of each tentative species (Euborellia sp. 1, 2, or 3) and E. femoralis form monophyletic clades with 100% support. Interestingly, the clade of Euborellia sp.1 (from Malaysia) consisted of two subclades, one of which also included E. annulata from the West Indies. The sister relationship between Euborellia sp. 3 and E. femoralis was also supported with high confidence (99%). Euborellia sp. (China) and Euborellia plebeja (Dohrn, 1863), for which only the fully winged form has been reported (except for records of those treated as Euborellia sp. 3 here), form a clade with 91% support, with its sister place being Euborellia sp. 2 (95% support). Placement of E. annulipes in this genus was not settled.
After the description of Forficula annulata from “Americae meridionalis” (= southern America) by
Interestingly, our Euborellia sp. 1 made a cluster, both in morphology (Fig.
Interestingly, our molecular analysis detected two sub-clades in Euborellia annulata: a female from Bayan Lepas, Penang Island, Malaysia (LC715987) showed only 1.8% divergence from a female from the West Indies (LC740580), while the other Malaysian samples (LC715988–LC715990), including those from other sites of Penang Island, were clustered with 4.8–6.5% differences from the formers (Table
In addition to Euborellia sp. 2, two macropterous Euborellia species, E. plebeja and E. femoralis, have been reported from the Orient (
The present results clearly show that Euborellia sp. 3 of the main islands of Japan (Honshu, Shikoku, and Kyushu) is a distinct species. After the proposed (and erroneous) synonymy of E. minuta and E. stali as E. plebeja (
Diagnostic features of the three brachypterous Euborellia species from East and Southeast Asia. Female E. annulata and E. pallipes are difficult to distinguish, but the former is usually smaller (Fig.
Traits | Euborellia annulata (Euborellia sp. 1) | Euborellia philippinensis (Euborellia sp. 2) | Euborellia pallipes (Euborellia sp. 3) |
---|---|---|---|
Black markings of legs | Markings of mid femur are darker than those of basal half of tibia | Markings of mid femur are weaker than those of basal half of tibia | In almost same intensity |
Lateral sides of male abdominal segments 6th (or 7th) to 9th | Carinated | Not carinated | Carinated |
Outer margin of parameres | Not strongly angular, rounded | Strongly angular | Not strongly angular, rounded |
Y-shaped area of pigmentation on penis lobes | Present | Absent | Absent |
Interestingly, our molecular analysis revealed that this species is sister to E. femoralis (Fig.
We thank H.-S. Tee, L.-H. Ang, C.-C. Lee, and X.-Y. Goh for assistance in collecting specimens in the field, and C. Girod and N. Moulin for providing us with important material collected in an inventory for the Conservatoire du Littoral (French coastal protection agency). We also thank W.-J. Wu, C.-C. Scotty Yang, H. Karube, and K. Watanabe for their assistance with examining specimens preserved in
The samples and results of the crossing experiments
Data type: table (Pdf file)
The specimens and results of morphological measurements
Data type: table (Pdf file)
Species and the collection sites of the Dermaptera samples sequenced in this study
Data type: table (Pdf file)