Research Article |
Corresponding author: Boonsatien Boonsoong ( fscibtb@ku.ac.th ) Academic editor: Lyndall Pereira-da-Conceicoa
© 2022 Chonlakran Auychinda, Michel Sartori, Boonsatien Boonsoong.
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:
Auychinda C, Sartori M, Boonsoong B (2022) Cincticostella ebura sp. nov., a new species of mayfly (Ephemeroptera, Ephemerellidae) from Thailand. ZooKeys 1130: 191-204. https://doi.org/10.3897/zookeys.1130.91039
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A new species of ephemerellid mayfly, Cincticostella ebura sp. nov., is described based on larvae collected in a stream from Nan Province, Thailand. This new species is classified in the nigra complex of the genus Cincticostella based on morphological and COI phylogeny evidence. The new species is closely related to C. nigra (Uéno, 1928) and C. funki Martynov, Selvakumar, Palatov & Vasanth, 2021 based on body colour pattern. Investigation of the chorionic structure of the new species showed similar details to those of other species within this species complex. The phylogeny also placed this species into a monophyletic group with C. nigra (Uéno, 1928), C. elongatula (McLachlan, 1875) and C. fusca Kang & Yang, 1995.
COI, ephemerellid mayfly, insolta complex, integrative taxonomy, nigra complex
The genus Cincticostella was established by
Recently,
Currently, 22 species are recognised in the world, of which 17 are found in the Oriental region (
In 2019, we collected larval material from Nan Province, Thailand. These specimens were morphologically identified and were found to share many characters with the East Palearctic species, C. nigra (Uéno, 1928) and Oriental species, C. funki Martynov, Selvakumar, Palatov & Vasanth, 2021. However, some characters were different and together with the distinct geographic or ecological distribution, we therefore classified these specimens as a new species and the first recorded species of the nigra complex in Thailand. The morphological characters of the mature larvae are described, including the chorionic structures, which were investigated by scanning electron microscopy (SEM). In addition, the COI gene of the new species was sequenced and a phylogenetic tree was reconstructed using our sequences and some Cincticostella COI sequences available in the GenBank database. Species delimitation was also based on the genetic distances using Kimura 2-parameter (K2P) analysis (
Larvae were collected using a D-frame kick net in the riffles of fast-flowing areas. The specimens were preserved in 95% ethanol and a whole larva was selected and dissected for morphological observation. The morphological characters were observed by permanent slide preparation using Euparal as a medium and observed by light microscopy. The eggs were also dissected from a late female larva. The chorionic structure was investigated by drying the eggs, coating them with gold, and observing them by SEM with a FEI Quanta 450. Final plates were prepared with Adobe Photoshop® CC 2020. Holotype and paratype specimens of the new species are deposited in the collections of the Zoological Museum at Kasetsart University in Bangkok, Thailand [ZMKU] and the Museum of Zoology in Lausanne, Switzerland [
Thoracic muscles were dissected for DNA extraction. Total genomic DNA was extracted with a genomic DNA extraction kit (NucleoSpin, Macherey-Nagel, Germany) following the manufacturer’s protocol. The COI amplification was performed using LCO1490 and HCO2198 (
Family Ephemerellidae Klapálek, 1909
Holotype
: Male mature larva in ethanol, Thailand, Nan Province, Bo Kluea District, Mae Nam Wa stream, 19°16'22.6"N, 101°10'48.2"E, 848 m, 26.XI.2019, C. Auychinda leg. [ZMKU]. Paratypes: 30 larvae in ethanol, one on slide, same data as holotype [ZMKU]; 4 larvae in ethanol, same data as holotype [
Mature larva (in alcohol, Fig.
Head. Black without tubercles, prominent bright ocelli; antennae three times longer than head length. Labrum densely covered with long fine setae, apicolateral angles rounded; apicomedially with deep emargination; ratio of emargination length to maximum labrum length = 1: 4.7 (Fig.
Thorax. Black with distinct white median line. Pronotum rectangular without clear anterolateral projections. Mesonotum with rounded anterolateral projections, outer margins not notched (Fig.
Cincticostella ebura sp. nov. a thorax in dorsal view, a pair of tubercles was indicated by arrows b posterior projection of mesonotum c foreleg d foretarsal claw e mid-femur f hind-femur g abdominal terga V–VII h abdominal terga VIII–X i, j setae on apically dorsal forefemoral surface k setae on posterior margin of abdominal terga. Scale bars: 0.2 mm (a, b, c, e, f, g, h); 0.05 mm (d, j); 0.01 mm (k).
Abdomen. Terga I–X each with a pair of posteromedian projections, well developed into strong tubercles of terga IV–VIII (Figs
Eggs. Dissected from mature larva (Fig.
Adults. Unknown.
The pair of sub-median tubercles in the middle of mesonotum of early stages is prominent and variable in number, 2 or 4 tubercles (Fig.
The larva of Cincticostella ebura sp. nov. has a well-marked white median line along its body that can be used to separate it from other Cincticostella species. However, this pattern is also present in C. nigra (Uéno, 1928) and C. funki Martynov, Selvakumar, Palatov & Vasanth, 2021, and the body shape is quite similar (
The specific epithet ‘ebura’, which means ivory, is a reference to the pairs of long and curve tubercles on the abdominal posteromedian margins.
The type locality of Cincticostella ebura sp. nov. is the Mae Nam Wa stream, Nan Province, Thailand (Fig.
Nan Province, northern Thailand.
The Bayesian phylogenetic tree reconstruction of COI showed that Cincticostella forms a monophyletic lineage which is distinctly separated from the other ephemerellid mayflies, with high probability branch support (Fig.
Pairwise genetic distances (COI) between species of Cincticostella using the Kimura 2-parameter.
Species | K2P genetic distances | ||||||||
---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
1. C. ebura sp. nov. | |||||||||
2. C. nigra | 0.22 | ||||||||
3. C. elongatula | 0.24 | 0.15 | |||||||
4. C. levanidovae | 0.24 | 0.24 | 0.24 | ||||||
5. C. tornata | 0.23 | 0.25 | 0.25 | 0.26 | |||||
6. C. femorata | 0.23 | 0.24 | 0.24 | 0.20 | 0.16 | ||||
7. C. gosei | 0.23 | 0.21 | 0.25 | 0.23 | 0.22 | 0.23 | |||
8. C. insolta | 0.22 | 0.25 | 0.23 | 0.22 | 0.22 | 0.22 | 0.22 | ||
9. C. orientalis | 0.26 | 0.26 | 0.26 | 0.25 | 0.22 | 0.25 | 0.23 | 0.23 | |
10. C. fusca | 0.21 | 0.22 | 0.24 | 0.23 | 0.22 | 0.24 | 0.23 | 0.24 | 0.23 |
Bayesian inference of COI sequences of some ephemerellid mayflies including Teloganopsis, Torleya and Cincticostella with probability branch support and GenBank accession numbers, or BOLD numbers, in brackets. The color bars indicate the species complex of the genus Cincticostella. The first column (#1) is the species complex following
Our morphological evaluation of C. ebura sp. nov, especially body coloration, revealed some similarities with C. nigra and C. funki. However, these three species inhabit different geographic areas, as C. nigra is only reported from the East Palaearctic (
The egg chorionic structure shows a similar pattern to that of the other Cincticostella species, including C. levanidovae, C. elongatula, C. nigra, C. fusca, C. orientalis, C. colossa and C. femorata (
From our results, C. ebura sp. nov. belongs to the nigra complex according to
Our molecular results support the placement of C. ebura sp. nov. into the nigra complex. In addition, our analysis supports the placement of C. elongatula (McLachlan, 1875) by
In our reconstruction, C. orientalis (Tshernova, 1952) was recovered as the sister clade of all Cincticostella species, whereas
1 | Mid- and hind femora expanded; head with a pair of tubercles | 2 |
– | Mid- and hind femora not expanded; head without tubercles | 3 |
2 | Pronotum with broad and extended anterolateral projection around head capsule | C. femorata |
– | Pronotum with moderately anterolateral projection… | C. insolta |
3 | Body black without median pale line; maxillary without palpi | C. gosei |
– | Body black with median pale line along the body; maxillary with three-segmented palpi | C. ebura sp. nov. |
1 | Chorion covered with broken reticulation (Ishiwata, 2003, figs 7, 8) | C. levanidovae |
– | Chorion covered with not broken reticulation | 2 |
2 | Chorionic surface with one tubercle (rarely two) at the centre of hexagonal ridge | 3 |
– | Chorionic surface with a variety of tubercles (1–5) at the centre of hexagonal ridge ( |
C. colossa , C. fusca , C. orientalis and C. ebura sp. nov. |
3 | Egg relatively large, surface seems to be rough (length 162–168 µm, width 116–120 µm) ( |
C. elongatula and C. nigra |
– | Egg relatively small, surface seems to be smooth (length 152 µm, width 114.6 µm) ( |
C. femorata |
This research has been supported by the Centre of Excellence on Biodiversity (BDC) Office of Higher Education Commission (BDC-PG2-161004). This research was approved by the Institutional Animal Care and Use Committee, Faculty of Science, Kasetsart University, Thailand under Project number ACKU61-SCI-028. We would like to thank Dr. Jean-Luc Gattolliat who provided us with the COI sequence of C. femorata in the difficult situation of the COVID-19 pandemic. We are most grateful to Dr. Alexander V. Martynov who provided good suggestions about the related species, Cincticostella funki also, our colleagues for their assistance during field trips. We would like to thank the Department of Zoology and the Faculty of Science at Kasetsart University in Bangkok for their assistance and use of their facilities.