A preliminary molecular phylogeny of the genus Scobura, with a synonym of Scobura masutaroi (Lepidoptera, Hesperiidae)

Abstract A molecular phylogeny of the genus Scobura based on the mitochondrial COI and the nuclear EF-1α genes using maximum likelihood and Bayesian inference is proposed. The analyses include 19 specimens from nine ingroup species. The monophyly of Scobura is not strongly supported, but two strongly supported monophyletic groups within the genus are recognized: the Scobura coniata group and the Scobura woolletti group. Judging from combination of the molecular evidence and morphological features, the former consists of six species, including Scobura masutaroi, while four species belong to the latter. Scobura mouchai Krajcik, 2013 is confirmed to be a syn. n. of Scobura masutaroi Sugiyama, 1996. The key to the species of the genus Scobura is modified to reflect these results.


Introduction
The skipper genus Scobura Elwes & Edwards, 1897 was recently revised by Fan et al. (2010), who recognized 14 species. The genus Scobura, however, includes another species, S. masutaroi, Sugiyama 1996. Fan et al. (2010 overlooked the existence of this taxon and did not include it in their revisional work, which resulted in Krajcik (2013) proposing a new taxon, S. mouchai, from Shaanxi.
Although a comprehensive morphological revision of the genus has been completed, no phylogenetic analysis has been performed to infer relationships within the genus. In the present study, we present a preliminary phylogeny of Scobura, based on molecular evidence. By comparing molecular and morphological evidence, we examine whether S. mouchai is a synonym of S. masutaroi.

Morphological examination
See Fan et al. (2010) for materials for the morphological study. In order to examine the wing venation, wings were removed from thorax, cleaned with 95% ethanol, and dyed red with acetocarmine (Wang et al. 2011).

Taxon sampling
Twenty-three specimens including nine of the 15 valid species of Scobura and four outgroup species were included in the phylogenetic reconstruction. Detailed information on the specimens is provided in Table 1. Specimens used in this study were mainly deposited in the Insect Collection, Department of Entomology, South China Agriculture University (SCAU), except for some specimens in Kyushu University museum (KU) and Mr. Hiroaki Onodera's private collection.

Laboratory protocols
Genomic DNA was extracted from the thorax of specimens preserved in ethanol, or from legs of dried specimens, using Magen's Blood/cell/tissue DNA extraction kit. One mitochondrial gene cytochrome c oxidase I (COI) and one nuclear gene elongation factor 1-α (EF-1α) were used as molecular phylogenetic markers. The following primers were used for amplification and sequencing in this study: for COI -primers LCO1490 and HCO2198 (Folmer et al. 1994); for EF-1α -primers ef44 and efrcM4 (Monteiro and Pierce 2001). Ploymerase Chain Reaction (PCR) were performed in 20 µl volumes containing 1 µl Amplified DNA products were purified using an Agarose Gel Extraction kit (Magen Biotech), and directly sequenced, or cloned with pMD18-T vector (Takara Inc), and then sequenced. Sequencing was performed using the ABI 3730 automated sequencer. All sequences were submitted to the Genbank database (accession numbers are given in Table1).

Phylogenetic analyses
Alignment of the DNA sequences were performed in Clustal X (Thompson et al. 1997) and edited manually in MEGA 6.0 (Tamura et al. 2013). All base frequencies and molecular character statistics were calculated in MEGA 6.0. Phylogenetic trees were constructed under maximum Likelihood (ML) and Bayesian inference (BI) criteria. For ML analysis, RAxML version 8 (Stamatakis et al. 2014) was used on a concatenated data set of two genes, with 1000 rapid bootstrap replicates using GTR+G substitution model on the CIPRES Science Gateway (Miller et al. 2010). BI was carried out using Markov Chain Monte Carlo (MCMC) randomization in MrBayes v3.2.3 (Ronquist et al. 2012). We used reversiblejump MCMC to allow for sampling across the entire substitution rate models. Four Markov chains (three heated chains, one cold) were run for 500, 000 generations, with the first 25% of sampled trees discarded as burn-in. The two independent runs were considered to have converged when the standard deviation of split frequencies value was <0.01. The convergence of the analysis was determined in Tracer v1.6 (Rambaut et al. 2014). Bayesian posterior probabilities (PP) and ML bootstrap values (BP) were used to evaluate branch support.

Sequence data
From a total of 23 samples, 22 sequences for COI and 21 for EF-1α were obtained. The alignment of the combined sequences consisted of a total of 1724 bp (658 bp of COI and 1066 bp of EF-1α genes, respectively), including 277 variable and 200 informative sites.

Phylogenetic analyses
The two model-based analyses (BI and ML) revealed nearly identical topologies, differing mainly in branch support (Fig. 1). In both analyses, the monophyly of the genus Scobura is weakly supported (BP = 44, PP = 0.87). Within the genus, although support for the basal clades was low, the Scobura species included here are clearly distinguished from each other, and formed four clades: the S. isota clade (which only included two representative specimens), Clade A, the S. cephaloides clade (only with two representative specimens), and Clade B. Clade A is comprised by S. stellata + (S. parawoolletti + S. woolletti) and receive high bootstrap support and posterior probability (BP = 99, PP = 1.00). We hereafter called the clade S. woolletti group. In all the analyses, S. cephaloides is sister to Clade B, with moderate support (BP = 63, PP = 0.92), whereas the relationships between S. isota and the other clades (Clade A, S. cephaloides and Clade B) remain unresolved.

Discussion
Although our phylogenetic analyses do not strongly support the monophyly of the genus Scobura, two strongly supported monophyletic groups within the genus are recognized: the S. coniata group and the S. woolletti group. The members of the coniata group share the following four morphological characters: 1) male band of scent scales on both sides of veins CuA 1 and CuA 2 and above 2A on the forewing (Fig. 2); 2) juxta U-shaped with two spine bearing arms, flat at base; 3) tegumen without socius; and 4) uncus thin and long. S. masutaroi is nested within this group. In our present analyses, two individuals (He 300, 301) of masutaroi from Nibashan, Sichuan (close to Dujiangyan, Sichuan, the type locality of S. masutaroi) and an individual (He303) from Jialingjiang, Fengxian, Shaanxi (the type locality of S. mouchai) are clearly grouped together with strong support values (BP = 100, PP = 1.00). Moreover, the pairwise P2K distances in COI between the species in the S. coniata group range from 3.3% to 6.1% with divergences between species averaging 4.5%, while divergence between individuals of S. masutaroi from Sichuan and Shaanxi province was 0.2%.
Based on the original description, distribution data, and the illustrations provided by Krajcik (2013), as well as our phylogenetic inferences, we conclude that S. mouchai is identical to S. masutaroi and should be considered a junior synonym. The male genitalia are illustrated herein, and the female genitalia are described for the first time. On the basis of morphological study (Devyatkin, 2004), two other species, S. phuongi and S. evani, which are not included in the present study, likely also belong to this group.
A well-support clade comprised by S. stellata, S. parawoolletti and S. woolletti was recovered in all analyses. These species share the following three characters: 1) hindwing with white spots on underside but not on upperside; 2) socius slender and pointed at tip; and 3) juxta funnel-like, thin and long basally. The generic name Mimambrix Riley, 1923 was proposed with Mimambrix woolletti as the type species, but later synonymized by Evans (1949). We follow Evans' treatment and consider this clade as a species group within the genus Scobura. Based on morphological characters, the group also includes S. tytleri (Evans, 1914).

Taxonomic account
The key given by Fan et al. (2010) is modified to include S. masutaroi. The couplets leading to S. masuataroi only are included here. Couplets beyond 11 in the original increase their number by one. Diagnosis. Forewing length 17-18 mm. This species is different from other species of S. coniata group in the appearance of the wing upper side: forewing with yellow streak in subcosta space basally, a big cell spots solid across cell, the spot in space CuA 2 yellow; hindwing with spots in spaces CuA 1 and M 1 -M 2 yellow. Wing under side: forewing costal and submarginal spots yellow; hindwing all veins and submarginal spots from spaces Sc+R 1 to CuA 2 yellow; and all yellow submarginal spots conjoined both forewing and hindwing.   Sugiyama, 1996. (Sichuan). A Genitalia ring, lateral view; B aedeagus and juxta. C valva, inner view; D tegument, dorsal view.  Sugiyama, 1996 (Sichuan) Description. Male genitalia (Fig. 4): Tegumen without socius, weakly rounded from lateral view; uncus slender and much longer than tegumen; valva with transtilla rounded and sclerotized with small spines, ventro-distal process irregularly shaped with outer edge rounded, inner edge uneven, and distal part rectangular with densely small spines; saccus short and broad; gnathos absent; juxta U-shaped with two arms with densely spines.