A new species of alpine Apenetretus Kurnakov from Taiwan: evidences from DNA barcodes and morphological characteristics (Coleoptera, Carabidae, Patrobini)

Abstract 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 Apenetretus yushanensis Habu, Apenetretus nanhutanus Habu, and Apenetretus 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 Apenetretus 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 Apenetretus hsueshanensis to its sister group was dated to 0.94 mya.


Introduction
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 (Habu 1972). Ten species of the Epaphiopsis Ueno, a genus endemic to Taiwan, are found in the high altitude mountain ranges across Taiwan (Ueno 1989). In addition, the aforementioned species are either allopatrically distributed in specific mountains or have topography-matched divergences. Obviously, these divergent events are highly associated with the effect of mountain isolation.
The genus Apenetretus in Taiwan includes three described species, all of which inhabit alpine areas of different mountain ranges (Habu and Baba 1960;Löbl and Smetana 2003;Terada 2006). Apenetretus yushanensis (Habu, 1973) and A. nanhutanus (Habu, 1973) were first collected and described from Yushan and Nanhudashan, respectively (Habu 1973) (Fig. 1). A third species, A. smetanai (Zamotajlov & Sciaky, 1996), was collected by A. Smetana in Mt. Nenggaoshan in 1992. In the original description, A. yushanensis and A. nanhutanus were considered as members of Patrobus Dejean, 1821 and Apatrobus was considered a subgenus under Patrobus. In 1992, Zamotajlov proposed that Apatrobus be given genus status with the rationale that members of Apatrobus had both larger eyes and more prominent temples which were sub-equal in length with eyes and therefore distinctly different from species of Patrobus. Therefore, based on this definition, Apenetretus yushanensis and Apenetretus nanhutanus are moved from the genus Patrobus to Apatrobus, and the third species, Apenetretus semetanai (Zamotajlov & Sciaky, 1996), was published as Apatrobus semetanai as well. Subsequently, the phylogeny among taxa including genus Apatrobus was studied and the taxonomy of Apatrobus was rearranged accordingly (Zamotajlov 2002;Zamotajlov and Wrase 2006). Two subgenera of Apenetretus and Parapatrobus are apparently different from Apatrobus by the absence of setae on ventral side of claw segments thus the both genera were proposed to new sense as genera. The three species originally belong to the subgenus Apenetretus in Taiwan were consequently changed into Apenetretus yushanensis, Apenetretus nanhutanus, and Apenetretus smetanai, respectively (Zamotajlov 2002). Although Lorenz still treated Apentetretus as a subgenus of Apatrobus in the recent catalog (Lorenz 2005), we expediently follow the classification of Zamotajlov, using Apenetretus as the genus for the four species in this study.
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 (Habu 1973). As more specimens were collected, however, we found several stable characters, including male genital characters, which could be used to distinguish the Hsueshan specimens from the other Apenetretus species.
In order to further examine the morphologically similar species, molecular barcoding methods were utilized as a practical process to help reveal candidate cryptic species A. smetanai was collected in Hehuanshan; A. nanhutanus was collected in Nanhudashan; A. yushanensis was collected in Yushan. Area of elevation above 2,000 meters is shaded. among numerous unidentified taxa (Burns et al. 2008;Hebert et al. 2003b;Winterbottom et al. 2014;Yassin et al. 2008). Molecular clock method was also employed to analyze Apenetretus genetic divergence times. Here the morphological features of a new Apenetretus species are described, including a proposed phylogenetic relationship and divergence time with other species based on mitochondrial cytochrome c oxidase subunit I (COI) gene.

Study sites and sample collecting
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 (

Morphological measurements
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 (Hadley 2010). Characters were examined and measured with the use of ImageJ 1.48, image analyzing software (Schneider et al. 2012).

DNA extraction, amplification, and sequencing
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 BuccalAmp TM 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).

Molecular analyses and phylogeny reconstruction
Sequences were aligned with BioEdit 7.0 software (Hall 1999). Proportional distances among species were conducted using MEGA version 6 (Tamura et al. 2013). The optimal substitution model HKY+I was choice according to jModelTest for Maximum likelihood tree construction and molecular clock calculation (Darriba et al. 2012;Guindon and Gascuel 2003). Phylogenetic inference was performed using maximum likelihood (ML) method with 1,000 bootstrap replications with PhyML version 3.0 (Guindon et al. 2010). The strict molecular clock of the COI gene was calculated under the rate of 3.54% per million years with software BEAST version 1.8.0 (Drummond et al. 2012;Papadopoulou et al. 2010).  and a ratio of elytral length/width (EL/EW=1.76-1.90) that differs from all other species ( Fig. 2A) 1.67-1.75, 1.67, 1.53-1.67, respectively (Habu 1973;Zamotajlov and Sciaky 1996). This character is especially useful in separating male individuals. Male genitalia; aedeagus large (ca. 3 mm in length) and more slender than the other three species (ca. 2.5 mm in length); extremely elongated and twisted after middle (Figs 3,  4). Apical portion of the parameres is prolonged and longer than the other species (Fig.  5) (Habu 1973;Zamotajlov and Sciaky 1996).
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. 6A), some with only one anterior and one posterior (Fig. 6C), sometimes one between eyes and clypeus, one anterior, and one posterior (Fig. 6B), or one anterior, one between anterior and posterior, and one posterior (Fig. 6D); distance between supraorbital posterior setae rather short, 0.78 (0.73-0.84) times as wide as anterior seta distance; frontal impressions deep, reaching clypeal setae, sometimes divergent posteriorly as A. smetanai; third segment of antenna rather long, 1.47 (1.23-1.59) times as long as forth segment; forth segment of antenna longer than fifth segment, 1.09 (1.03-1.15) times as fifth segment; tenth segment 1.78 (1.68-1.94) times as long as wide; eleventh segment rather prolonged, 2.5 (2.29-2.77) times as long as wide.
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 (Habu 1973;Zamotajlov and Sciaky 1996), 1.82 (1.76-1.90) times as long as wide, widest behind middle, 1.42 (1.30-1.54) times as wide as pronotum, shoulders with one small tooth on each side, wider than posterior margin of pronotum; microsculpture distinct, isodiametric; lateral margin subsinuate before one third, then rounded, apex elongated subapically; striae rather shallow, sometimes finely punctate; scutellary striole punctate; intervals flat, 3 rd interval with 3 pores at 0.22 (0.20-0.26), 0.49 (0.41-0.52), and 0.73 (0.69-0.77) times of elytra length; marginal series composed of 10-12 pores. 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. 2B, C) slender, curved to right side in dorsal view, curved and elongate before middle (Fig. 2B); apical lamella extremely twisted toward right side, forming a ridge at middle in dorsal view and hammer shape at apex in lateral view (Fig.  2C); left margin reflexed and sinuate in dorsal view; parameres different in shape and size of left and right, left paramere wider than right one, apical projection extended, much longer than the other three species, apex with two long and one short setae, and two short setae at each subapical margin (Fig. 2D). Taiwan   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 ( 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. 5A, E)  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. 3A); apical portion of parameres markedly prolonged ( Fig. 5A

Genetic differentiation of Apenetretus in Taiwan
Phylogenetic analysis of molecular work with the COI gene (686 bp) shows four distinct lineages within the Apenetretus of Taiwan (Fig. 7). Apenetretus yushanensis is the most basal lineage; members of A. hsueshanensis form a sister group to members of A. nanhutanus and A. smetanai (Fig. 7). The tree topology is consistent with the results of genetic divergence which informs that the most distinct species is A. yushanensis and the least divergent species are A. smetanai and A. nanhutanus (Table 2). It is worth noting that the genetic p-distance among these Apenetretus species are close or higher than 2%, the value defined as the general threshold of species differentiation (Hebert et al.    Hebert et al. 2003b). The divergent trend among Apenetretus species is likely to fit with the geological topology of the mountain ranges in Taiwan, where Yushan and Hsueshan Ranges are distinct from Central Mountain Range including Hehuanshan and Nanhudashan. The southern A. yushanensis is the most divergent one to the other three northern Apenetretus at ca. 1.81 million years ago (mya). The divergence time between A. hsueshanensis and its sister group was dated to 0.94 mya, a period which is sufficient for speciation to occur (Fig. 8), which further supports our findings that there is an independent species occurring in Hsueshan. Therefore, the localized Hsuehsan carabids with >2% COI divergent content have most likely speciated allopatrically due to the effect of mountain-island isolation. Interestingly, the divergent time between A. smetanai and A. nanhutanus, the two most closely distributed and morphologically similar species appear to have diverged only 0.53 mya. It is yet unclear if there is a geographical barrier between two species, so the possibility is exit that the two species may have other forms of isolated barrier such as isolated by distance or intermittently contact due to glacial cycles. The question can be resolved only by examinations and analyses of series collection along Central Mountain Range.
portant 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)