Research Article |
Corresponding author: Wen-Bin Yeh ( wbyeh@nchu.edu.tw ) Academic editor: Achille Casale
© 2016 Yi-Ming Weng, Wen-Bin Yeh, Man-Miao Yang.
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:
Weng Y-M, Yeh W-B, Yang M-M (2016) A new species of alpine Apenetretus Kurnakov from Taiwan: evidences from DNA barcodes and morphological characteristics (Coleoptera, Carabidae, Patrobini). ZooKeys 584: 121-134. https://doi.org/10.3897/zookeys.584.6320
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There are three isolated mountain ranges in Taiwan including Hsueshan Range, Central Mountain Range, and Yushan Range. The rise of these mountains has resulted in the isolation of some species and caused allopatric distribution resulting in divergence and speciation events of high mountain carabids, especially the flightless carabids such as Epaphiopsis, Apenetretus, and partial Nebria. Genus Apenetretus Kurnakov (1960) is typically distributed in high mountain areas of Taiwan. Three of the currently known Apenetretus species have been described from different mountain ranges. These species include A. yushanensis Habu, A. nanhutanus Habu, and A. smetanai Zamotajlov and Sciaky. In this study, a new species is described from Hsueshan, a mountain separated from the ranges of the previous known species, Apenetretus hsueshanensis sp. n. A key to the Taiwanese Apenetretus is included. A reconstructed phylogeny of the Taiwanese Apenetretus is introduced with the use of mitochondrial cytochrome c oxidase subunit I (COI) gene. Molecular data and geographical distribution of Apenetretus support the morphological characteristics observed among those mountain-isolated species and confirms the new species as being distinctly different. Moreover, lineage calibration suggests that the southern A. yushanensis is the most distant one compared to the other three northern Apenetretus at ca. 1.81 million years ago (mya), while the divergence time of A. hsueshanensis to its sister group was dated to 0.94 mya.
Apenetretus , Carabidae , Hsueshan, mountain island isolation, new species
In Taiwan, mountain ranges that have become isolated over time have played a major role promoting divergent events of high mountain dwelling carabids, especially in species with flightless adults. For example, in Nebria formosana Habu and N. niitakana Kano, morphological variation has been described in populations across mountain ranges (
The genus Apenetretus in Taiwan includes three described species, all of which inhabit alpine areas of different mountain ranges (
According to Habu’s original description, one additional female specimen with larger body and longer, depressed elytra from Mt. Hsueshan (Mt. T’zu-kao) has been collected and was considered by him as a local variety of A. yushanensis (
In order to further examine the morphologically similar species, molecular barcoding methods were utilized as a practical process to help reveal candidate cryptic species among numerous unidentified taxa (
Specimens of Apenetretus were collected by hand from various alpine areas across Taiwan. Specimens from the three species preciously described were collected from their respective mountain ranges including A. yushanensis from Yushan, A. nanhutanus from Nanhudashan, and A. smetanai from near Nenggaoshan (Table
Sample location | latitude | longitude | elevation (m) | |
---|---|---|---|---|
A. hsueshanensis sp. n. | Hsueshan | 24°23.6N | 121°14.7E | 3,330 |
A. yushanensis | Yushan | 23°28.5N | 120°57.8E | 3,369 |
A. nanhutanus | Nanhutashan | 24°22.1N | 121°26.5E | 3,394 |
A. smetanai | Hehuanshan | 24°8.2N | 121°16.5E | 3,100 |
Measurements of morphological characters were done with a Leica S8APO microscope connected to a Canon 600D camera. After taking character photos, images were stacked with software CombineZP (
For molecular work, twelve individuals of A. hsueshanensis sp. n., ten of A. smetanai, ten of A. nanhutanus, and eight of A. yushanensis were used for DNA extraction. Following the instructions of BuccalAmpTM DNA Extraction Kit (Epicentre Biotechnologies, Madison, WI), genomic DNA was extracted from one hind tarsus of each individual by glass homogenizer grounding in 50 µl QuickExtract Solution, centrifuging for 15 sec, incubating at 65 °C for 10 min, centrifuging for 15 sec again, and then incubating at 98 °C for 2 min. Finally, the resultant genomic DNA products were stored at -20 °C for polymerase chain reaction (PCR).
Mitochondrial COI barcode region was amplified with forward primer Col46 (5’-AACCATAAAGATATTGGAAC-3’) and reverse primer Col731 (5’-CAACAT TTATTTTGATTTTTTGG-3’) in PCR (
Sequences were aligned with BioEdit 7.0 software (
Taiwan: Mt. Hsueshan, Hsei-Pa National Park, Black Forest near Sanliujiu Cabin, ca. 3,330 m elevation, 24°23.6N, 121°14.7E.
Holotype: a male, deposited in National Chung-Hsing University (
The new species is named after the original collecting locality, Mt. Hsueshan, where it is likely endemic.
Apenetretus hsueshanensis sp. n. is morphologically similar to the other Taiwanese Apenetretus species (A. yushanensis, A. nanhutanus, and A. smetanai). It can be distinguished externally from the other three species by having more slender elytra and a ratio of elytral length/width (EL/EW=1.76–1.90) that differs from all other species (Fig.
Male 10.79–11.77 mm in length, 3.50–3.79 mm in width, female 11.10–12.22 mm in length, 3.71–4.01 mm in width. Color brown to black, ventral surface reddish brown; labrum, mandibles, palpi, legs, and margin of pronotum and elytra lighter in color (Fig.
Head convex, frontal impression, neck-constriction punctate; microsculpture faint and isodiametric in dorsal view; neck-constriction deep; temporae faintly tumid, longer than eyes, 1.11 (0.88–1.25) times as long as eye in average (only one individual in fifteen individuals has longer eye length than temporae); eye large, convex; with tooth at subapical terminal; palpi truncate at apex; supraorbital setae varied, some individuals have two closely anterior and one posterior (Fig.
Variation in supraobital setae placement of Apenetretus hsueshanensis sp. n. A two close anterior setae and one posterior B one between eyes and clypeus, one anterior, and one posterior; C, one anterior and one posterior D one anterior, one between anterior and posterior, and one posterior. Scale bar: 1 mm.
Pronotum weakly convex, widest at about one third, 1.22 (1.18–1.32) times as wide as head, 1.23 (1.17–1.29) times as wide as long, 1.35 (1.28–1.39) times as wide as posterior margin, anterior generally as wide as posterior margin, 1.00 (0.95–1.06) times as anterior margin as posterior margin; microsculpture faint and isodiametric; anterior margin straight to rounded and protrudingt at angles; surface faintly punctate at apical areas, rather punctate along median line, lateral margins, and basal area; posterior margin straight, shallowly sinuate near hind angles; hind angles acute to rectangular, slightly prominent laterally; lateral margin subsinuate, from front angles to the widest points, rather round from the widest points to the turning points, then prominent to the posterior seta pore; anterior marginal setae located before the widest point; posterior setae in hind angles; median line deep, sometimes reaching both extremities, generally reaching to anterior transverse impression; anterior transverse impression shallow, sometimes deep and forming a Y-shaped impression; posterior impression and basal foveae deep; disk smooth, rather cordate.
Wings atrophied, 0.3 times as long as elytra; elytra rather convex, ovate and more slender than the other three species (
Mesepistern, metepistern, and mesostern, lateral of prostern, metasternum, and pregenital sterna 1 with distinct punctures; ventral side of neck constriction shallowly rugose on each side; metepistern longer than wide.
Aedeagus (Fig.
1 | Antenna moniliform, reaching to basal one seventh of elytra; apical part of parameres short, with one short seta at apex and one or no subapical seta (Fig. |
A. yushanensis Habu |
– | Antenna slender, reaching to basal one fifth to one sixth of elytra; apical part of parameres longer, with two long seta and one or no short seta at apex, and two short subapical seta on each side (Fig. |
2 |
2 | Elytra prolonged, more than one and three fourth as long as wide; aedeagus long, (~3mm), extended and extremely twisted toward right side behind middle (Fig. |
A. hsueshanensis sp. n. |
– | Elytra not prolonged, one and one half to one and three fourth as long as wide; aedeagus shorter, mostly 2–2.5 mm long, evenly contracted toward apex; apical part of parameres less prolonged (Fig. |
3 |
3 | Palpi truncate and depressed apically; temporae longer than eye; front angles of pronotum stronger projected | A. smetanai Zamotajlov & Sciaky |
– | Palpi not truncate; temporae same length as eye; anterior angles of pronotum weakly projected | A. nanhutanus Habu |
Phylogenetic analysis of molecular work with the COI gene (686 bp) shows four distinct lineages within the Apenetretus of Taiwan (Fig.
Molecular clock dating of mitochondrial COI gene with BEAST ver. 1.8.0. The oldest divergence between A. yushanensis and the other Apenetretus species occurred at 1.81 million years ago (mya); the divergence between A. hsueshanensis and the group of A. smetanai and A. nanhutanus occurred at 0.94 mya; and the divergence between A. smetanai and A. nanhutanus occurred at 0.53 mya.
We thank Wesley Hunting, who taught us techniques of collection, preparation and dissection of carabid beetles and provided assistance with an early version of this work. We thank Dr. Dave Kavanaugh, who taught us how to examine carabid genitalia in detail and understand important characters. We also thank Cheng-Lung Tsai, William Chiu, Zong-Han Yang, and Yi-Zhang Liao, who helped us collect the examined specimens from the high mountains of Taiwan. We thank Scotty Yang, who provided important support both materially and spiritually, and Shih-Tsai Yang who reviewed and corrected our species descriptions. The authors also acknowledge the High-throughput Genome and Big Data Analysis Core Facility, Taiwan (MOST 104-2319-B-010-001), for sequencing. This study was partially supported by the Shei-Pa National Park (SP10102)