Research Article |
Corresponding author: Michele Rossini ( micros.naturae@gmail.com ) Academic editor: Andrey Frolov
© 2021 Michele Rossini, Olivier Montreuil, Vasily Grebennikov, Sergei Tarasov.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Rossini M, Montreuil O, Grebennikov V, Tarasov S (2021) Genome sequencing reveals extraordinary cephalic horns in the Madagascan dung beetle genus Helictopleurus (Coleoptera, Scarabaeinae): insight from a revision of fungicola species group. ZooKeys 1033: 63-79. https://doi.org/10.3897/zookeys.1033.63527
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In this study, we test and corroborate the phylogenetic position of Heterosyphus within Helictopleurus using mitogenomes and nuclear loci. Our recent samplings revealed that males of the former Heterosyphus sicardi Paulian, 1975 (today under Helictopleurus d’Orbigny, 1915) have extraordinary bilateral clypeal horns which are exclusive within the genus. We provide a taxonomic review of the fungicola species group of Helictopleurus and discuss the systematic position of H. sicardi within the group. The male phenotype of H. sicardi is described and photographs of the body and genitalia of the members of the fungicola group are given, as well as a diagnostic key to species of the group. Helictopleurus fungicola peyrierasi is considered to be a distinct species within the genus (H. peyrierasi stat. rest.). Helictopleurus pluristriatus d’Orbigny, 1915 syn. nov. is established as a junior synonym of H. fungicola (Fairmaire, 1899).
dung beetles, fungicola species group, Heterosyphus, Madagascar, male horns, mitogenomics, Scarabaeinae
The dung beetle tribe Oniticellini (Coleoptera, Scarabaeinae) was represented by two endemic genera in Madagascar, namely Helictopleurus d’Orbigny, 1915 and Heterosyphus Paulian, 1975. With 68 known species and subspecies, Helictopleurus was thought to have males with only simple cylindrical horns or carina on the head, which are common across various dung beetle lineages. At the same time, the monotypic and extremely rare Heterosyphus was thought to be hornless. Previous phylogenetic analysis of Madagascan Oniticellini (
Helictopleurus sicardi, the former member of the monotypic genus Heterosyphus (
Thus, considering the exclusive phenotype of H. sicardi and its previous placement in a separate genus, we test the phylogenetic position of this species within Helictopleurus using mitogenomic data and nuclear loci. Our 19-gene phylogenetic analysis of Helictopleurus and other genera from the tribe Oniticellini corroborates the results of
Voucher specimens and type material analyzed throughout the study are deposited in the following institutes:
MZHF Finnish Zoology Museum of Natural History (LUOMUS), Helsinki (S. Tarasov, J. Mattila).
The external morphology, along with the anatomy of the male and female genitalia of a total of 39 Helictopleurus species currently assigned to seven of the nine species groups (
Morphological study was performed under a Leica S9D stereomicroscope. Habitus photographs were taken with a Canon EOS 5D camera and a Canon MP-E 65mm, f/2.8, 1–5× macro lens, using the Cognisys Stackshot automated system; male genitalia were photographed with a Nikon SMZ25 stereomicroscope coupled with a DS-Ri2 camera. Zerene Stacker (v. 1.04 Build T2020-05-22-1330) software and NIS-Elements-BR (Nikon Imaging Software Basic Research) were used to process and combine multiple photographs. Images were enhanced and arranged in plates in Adobe Photoshop and Illustrator CC 2015.
Genomic DNA was extracted from an ethanol-preserved female of H. sicardi (http://id.luomus.fi/NC.03) following the Qiagen DNeasy Blood & Tissue Kit (QIAGEN). The quality control was performed with a Qubit dsDNA HS (Invitrogen) and Fragment Analyzer (AATI). The generated Nextera Flex library (Illumina) was sequenced using Illumina NextSeq 500 sequencer with the cycles 170-8-8-132 that yielded the lowest coverage genome of H. sicardi.
The read quality was checked with FastQC (
The ingroup consisted of 44 operational taxonomic units (OTUs) belonging to ~30 species of Helictopleurus; two H. sicardi OTUs were used, the new one and the one from previous phylogenetic study (
Gene fragments were individually aligned using MAFFT (
The combined phylogenetic analyses of the fragments of 19 mitochondrial and nuclear genes support the monophyly of the genus Helictopleurus (BS 83) (Fig.
Molecular phylogenies and morphological synapomorphies of the fungicola species group A phylogenetic position of H. sicardi within Helictopleurus. Helictopleurus neoamplicollis and H. carbonarius are highlighted with colored dashed line to indicate their previous placement B COI-based phylogeny: magnification of the fungicola clade and phylogenetic position of H. peyrierasi stat. rest. C synapomorphies of the fungicola species group: basal pygidial ridge (BPyR); mesotarsal teeth (MsT) on the first tarsomere; parameres (P) elongated and evenly curved downward, basoventral lobes of the parameres bell shaped (BVLb); superior side of the membranous sac of the endophallus with spines (EpS); lamella copulatrix very simple (LC) D detail of the clypeal horns of the male of H. sicardi.
Interestingly, our combined analysis and that of
According to
These putative synapomorphies are not found in H. nigritulus or in H. neoamplicollis, which were formerly assigned to the fungicola group. Helictopleurus nigritulus exhibits characters that suggest its membership in the semivirens group (e.g., pronotum clearly larger than elytral width, surface of the body polished, with very shallow punctures, and head of the female ogive-shaped, with a transverse and straight carina in the frontoclypeal region). The correct taxonomic placement of H. neoamplicollis needs further investigation. Helictopleurus pluristriatus is here considered to be a new synonym of H. fungicola (see below).
Interestingly, H. villiersi Paulian & Cambefort, 1984, which was assigned to the viridiflavus group (
1 | Pronotal punctation strong and coarse; elytral interstriae granulose; major male with long and widely curved clypeal horns (Fig. |
H. sicardi (Paulian, 1975) |
– | Pronotal punctation very fine; elytral interstriae without granules; male without horns | 2 |
2 | Clypeal margin of male and female with two acute teeth at middle; male with a small transversal clypeal carina (Fig. |
H. viettei Paulian & Cambefort, 1984 |
– | Clypeal margin of male and female with three blunt to acute teeth at middle; male without clypeal carina | 3 |
3 | Frontoclypeal region with a distinct hump, postoccipital margin with a pointed tubercle in the middle (Fig. |
H. peyrierasi Paulian & Cambefort, 1984 stat. rest. |
– | Frontoclypeal region and postoccipital margin simple to slightly swollen (Fig. |
H. fungicola (Fairmaire, 1899) |
Oniticellus fungicola Fairmaire, 1899: 519.
Helictopleurus fungicola: d’Orbigny, 1915: 425;
Helictopleurus pluristriatus
d’Orbigny, 1915: 426 (syn. nov.);
Of H. fungicola: lectotype, male (here designated): “Madagr Suberblle H. Perrier / Muséum Paris Madagascar Perrier de la Bathie Coll. L. Fairmaire 1906 / TYPE / Oniticellus fungicola Frm Madag / Oniticellus fungicola Fairmaire, 1899 Rossini et al. des. 2021 / LECTOTYPE / Helictopleurus fungicola (Fairmaire, 1899) Rossini et al. det. 2021” (
Helictopleurus fungicola A, B habitus of male (A) and female (B) C, D lateral and anterior view of the aedeagus; basoventral lobes (BVLb) E endophallus: lamella copulatrix (LC) K, L details of the superior side of the membranous sac of the endophallus; Endophallic spines (EpS) O lateral view of the head: arrows indicating the absence of humps and tubercles. Helictopleurus peyrierasi stat. rest. F, G habitus of male (F) and female (G) H, I lateral and anterior view of the aedeagus J endophallus: superior (SpLC) and inferior (IpLC) part of the lamella copulatrix M, N details of the superior side of the membranous sac of the endophallus P lateral view of the head: arrows indicate the frontoclypeal hump and the postoccipital central tubercle.
Of H. pluristriatus: holotype, male: “Muséum Paris Madagascar Expéd. La Bonite, Gaudichaud 1837 / pluristriatus n. sp. d’Orb. / HOLOTYPE” (
This species is distributed from the northernmost region of Madagascar to the central-western coast. It is known from the Diana, Melaky, Boeny, and Menabe regions.
The examination of the holotype of H. pluristriatus (Fig.
Helictopleurus viettei A, B habitus of male (A) and female (B) C, D lateral and anterior view of the aedeagus; basoventral lobes (BVLb) E endophallus: endophallic spines (EpS); superior (SpLC) and inferior (IpLC) part of the lamella copulatrix. Helictopleurus pluristriatus F dorsal habitus of the holotype G lateral view of the aedeagus.
Helictopleurus peyrierasi
Paulian &
Helictopleurus fungicola peyrierasi:
Holotype, male: “Madagascar Ouest, réserve spéciale du Zombitsy, Est de Sakaraha, matsabory, 640m, 7-10.II.1974, P. Viette et A. Peyrieras / Holotype Helictopleurus peyrierasi n. sp. R. Paulian et Y. Cambefort det. 1991 / HOLOTYPE” (
Paratype, female: same data as holotype, except the collection date: “13.II.1974” (
This species is known from the central-western coast of Madagascar (Boeny and Menabe regions).
Helictopleurus viettei
Paulian & Cambefort 1984: 113;
Holotype, female: “Madagascar Ouest, réserve spéciale du Zombitsy, Est de Sakaraha, matsabory, 640m, 13.II.1974, P. viette et A. Peyrieras / Holotype Helictopleurus viettei n. sp. R. Paulian et Y. Cambefort det. 1984 / HOLOTYPE” (
Paratype, female: same data as holotype, except the collection date: “7–10.II.1974” (
Only known from south-western Madagascar (Atsimo-Andrefana region).
Heterosyphus sicardi
Paulian, 1975: 248;
Helictopleurus sicardi: Philips, 2016: 11, 13, 40–41 (synonymy Heterosyphus = Helictopleurus).
Lectotype, female (here designated): “Montagne d’Ambre. I. / Epactoides nar? / TYPE / Heterosyphus sicardi n.g. n.sp. R. Paulian det. /. Heterosyphus sicardi Paulian, 1975 des. Rossini et al. 2021 / LECTOTYPE. Helictopleurus sicardi (Paulian, 1975) Rossini et al. des. 2021” (
Paralectoype, female: “Antsiranana / Madagascar Montagne d’Ambre Muséum Paris Coll. Sicard 1930” (
Madagascar: “Mt. d’Ambre. −12.5281, 49.1709. 1080m. 1.i.2019. sift. MD31. V. Grebennikov, http://id.luomus.fi/NC.01” (1 male MZHF); same data, http://id.luomus.fi/NC.02 (1 female, MZHF; body parts disarticulated); same data, http://id.luomus.fi/NC.03 (1 female MZHF; body parts disarticulated, DNA material); same data (3 males, 7 females, MZHF); “Montagne d´Ambre. Jan 2004. Wet forest. Alt. 1300 m. fish baited trap. Iikka Hanski leg. / http://id.luomus.fi/GZ.19901. I.2004” (1 female, MZHF); same data, “http://id.luomus.fi/GZ.19902. I.2004” (1 male, MZHF).
Within the endemic Madagascar genus Helictopleurus, H. sicardi shares a series of morphological characters with the species here assigned to the fungicola group. These characters are the posterolateral margin of the pronotum extended in the propleural region with a short ridge beneath the lateral edge of the pronotum; clypeal margin of female with acute teeth at middle (three teeth as in H. fungicola and H. peyrierasi); parameres elongated; lamella copulatrix very simple and composed by a superior and inferior leaf-like parts; and superior side of the membranous sac of the endophallus with regions covered by scale-like spines. However, H. sicardi is easily distinguished from the other members of the fungicola group by the large punctation on the pronotum (very fine to absent in H. fungicola, H. peyrierasi, and H. viettei); male with a couple of long and widely curved clypeal horns (head unarmed in the remaining species; with an acute to obtuse post-occipital tubercle in H. peyrierasi); body completely brown (pronotum and head dark with blue to emerald green sheen, and elytra bicolored with reddish or yellow spots in the other species of the group); and elytra with rows of bright, elongated granules (granules absent in H. fungicola, H. peyrierasi, and H. viettei).
Body length and color. Body length from clypeal margin to elytral apices 7 mm, dorsal and ventral side of the body brown and bright, dorsal tegument clearly sericeous on the disc of the head, posteromedian region of pronotum and elytral interstriae; mouthparts and antennae light brown, setae light yellow to brownish.
Head. Clypeus with margin widely and evenly curved, with sides straight and parallel in proximity of the horns, genal margin curved, clypeogenal junction indicated by a short and shallow ridge, and by the base of the clypeal horns. Clypeus with two long and widely curved horns (Fig.
Helictopleurus sicardi A, B major (A) and minor (B) male dorsal habitus C major male lateral habitus D female dorsal habitus (lectotype) and original labels E propleural groove (PpG) F VIII interstrial ridge (IR) G, H lateral and anterior view of the aedeagus; basoventral lobes (BVLb) I endophallus: endophallic spines (EpS); superior (SpLC) and inferior (IpLC) part of the lamella copulatrix.
Thorax. In dorsal view, pronotum narrower with respect to elytra, in lateral view slightly convex. Lateral edges rather straight and weakly divergent from posterior angles to the middle, feebly concave and sinuate from middle to anterior angles. Pronotal anterior angles narrow and obtusely acuminate. Lateral and anterior pronotal edges complete and finely margined, posterior edge with no margin. The pronotal posterior edge is extended in the propleura, beneath the lateral edge of the pronotum, with a shallow groove (Fig.
Posteromedial pronotal region with punctation rugulose-lacunose and coarse, central and medial region without punctures, punctation more spaced in the anterior region. Several punctures associated with short and stout setae. Pronotal tegument shiny and smooth on anterior half, posterior half finely microsculptured and especially in the middle.
Propleuron weakly excavated at the bottom of the pronotal anterior angles; propleuron with two carinae, the internal carina thinner and straight, external one stronger and widely sinuate.
Episternum very narrow; mesosternal surface covered by coarse and dense punctures associated with short and stout setae, anterior region of mesosternum with a wide and smooth bead, triangularly pointed backward at middle; metasternum wide and steeply elevated with respect to the mesosternum in its superior region, surface smooth, with fine punctures on the disc and coarse punctures near the mesocoxae.
Abdomen. Elytra with eight glossy striae interrupted by a series of well-spaced and shallow punctures. Interstriae flat, with surface completely microreticulated, interstriae III–VI clearly swollen apically (carinated), interstria VIII with a longitudinal, thin ridge on basal one third (Fig.
Sternites ventrally visible, anterior margin with a double row of coarse and shallow punctures not perfectly aligned, three rows of punctures on the lateral most region of each sternite.
Pygidium flattened, completely margined, pygidial surface finely microreticulated and with scattered, shallow punctures.
Legs. Lateral margin of protibiae with four acute teeth distributed along the anterior half, posterior half serrated, apical and internal margin of protibia with an acute spur directed forward and slightly bent downward; ventral side of protibial with a longitudinal ridge that terminates apically with an acute tooth beneath the superior spur. Meso and metatibiae slender, enlarged apically, and respectively with two and one spiniform spurs at the apex. Profemora elongated, dorsal side smooth, ventral side with coarse and shallow punctures mostly concentrated on posterior half. Mesofemora and metafemora very slender and swollen posteriorly at middle, ventral surface with coarse and shallow punctures on posterior half, fine punctures anteriorly.
First segment of mesotarsi with four spine-like teeth in the external margin, two to three yellow setae inserted between each tooth.
Morphological variation. Minor males either with two small and straight clypeal horns that arise from the sides (Fig.
Females differ from males by the clypeal margin with three teeth distinctly reflexed upward, the lateral teeth obtuse, while the central one more acuminated, posterior margin of the lateral teeth with short setae; head surface covered by coarse and shallow punctures even on the clypeus where the punctation is shallower; pronotum not depressed medially; protibia without internoapical tooth; last abdominal sternite narrower at middle.
Male genitalia. Parameres elongated, ventrally defined by two straight laminas, basoventral side of the parameres with two lateral lobes obtusely squared (BVLb, Fig.
We are thankful to Gunilla Ståhls-Mäkelä for DNA extraction of H. sicardi, and to Alexey Solodovnikov (Natural History Museum of Denmark) and Victoria Twort (Finnish Museum of Natural History, LUOMUS) for their valuable advices. Tiana Vololontiana (Antananarivo, Madagascar) facilitated Madagascar fieldwork (249/18/MEEF/SG/DGF/DSAP/SCB) resulting in discovery of the horned male of Helictopleurus sicardi. Open access funded by Helsinki University Library.
This study was supported by the Pentti Tuomikoski Fund and the Academy of Finland Grant (#331631), awarded to ST.
DNA sequence alignment of the material examined in our study
Authors: Michele Rossini, Olivier Montreuil, Vasily Grebennikov, Sergei Tarasov
Data type: molecular data
Explanation note: Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.