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Research Article
A preliminary molecular phylogeny of the genus Scobura, with a synonym of Scobura masutaroi (Lepidoptera, Hesperiidae)
expand article infoZhen-Fu Huang, Wei Fei, Min Wang, Hideyuki Chiba§, Xiao-Ling Fan
‡ South China Agricultural University, Guangzhou, China
§ Bishop Museum, Honolulu, United States of America
Open Access

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 S. coniata group and the S. woolletti group. Judging from combination of the molecular evidence and morphological features, the former consists of six species, including S. masutaroi, while four species belong to the latter. S. mouchai Krajcik, 2013 is confirmed to be a syn. n. of S. masutaroi Sugiyama, 1996. The key to the species of the genus Scobura is modified to reflect these results.

Keywords

COI, EF-1α, Scobura masutaroi, Scobura mouchai

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.

Methods

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.

Voucher information and GenBank accession numbers for the specimens in this study.

Species Locality Latitude Longitude Voucher Number COI EF-1α
Scobura cephaloides kinkaEvans, 1949 China: Hainan 19.02N 109.53E SCAU He102 KY049936 KY049958
Scobura cephaloides kinkaEvans, 1949 Laos: Luang Prabang 19.93N 102.07E Onodera He553 KY049937 KY049959
Scobura coniata Hering, 1918 China: Guangdong 24.91N 113.04E SCAU He073 KY049938 KY049960
Scobura coniata Hering, 1918 China: Guangdong 24.87N 113.03E SCAU He472 KY049939 KY049961
Scobura hainana (Gu & Wang, 1997) China: Guangdong 24.87N 113.04E SCAU He471 KY049940 KY049962
Scobura hainana (Gu & Wang, 1997) China: Guangdong 24.87N 113.04E SCAU He487 KY049941 KY049963
Scobura hainana (Gu & Wang, 1997) China: Guangdong 24.87N 113.04E SCAU He488 KY049942 KY049964
Scobura isota (Swinhoe, 1893) Thailand: Kanchanaburi 14.08N 99.36E SCAU He538 KY049943 KY049965
Scobura isota (Swinhoe, 1893) Thailand: Mae Hong Son 19.35N 98.14E SCAU He468 KY049944 KY049966
Scobura lyso (Evans, 1939) China: Zhejiang 30.15N 119.25E SCAU He465 KY049945
Scobura lyso (Evans, 1939) China: Zhejiang 30.15N 119.25E SCAU He475 KY049946
Scobura masutaroi Sugiyama, 1996 China: Sichuan 29.94N 102.48E SCAU He300 KY049947 KY049967
Scobura masutaroi Sugiyama, 1996 China: Sichuan 29.94N 102.48E SCAU He301 KY049948 KY049968
Scobura masutaroi Sugiyama, 1996 (=mouchai) China: Shaanxi 31.91N 106.34E SCAU He303 KY049949 KY049969
Scobura parawoolletti Fan et al., 2010 China: Hainan 19.03N 109.53E SCAU He116 KY049950 KY049970
Scobura stellata Fan et al., 2010 China: Guangdong 24.92N 113.01E SCAU He036 KY049951 KY049971
Scobura woolletti (Riley, 1923) Indonesia: Kabandungan 6.77 S 106.60E KU He535 KY049952 KY049972
Scobura woolletti (Riley, 1923) Indonesia: Kabandungan 6.77 S 106.60E KU He536 KY049953 KY049973
Scobura woolletti (Riley, 1923) Indonesia: Kabandungan 6.77 S 106.60E KU He537 KY049954 KY049974
Suastus gremius (Fabricius, 1798) China: Guangdong 23.15N 113.34E SCAU He157 KY049955 KY049975
Suada swerga (deNicéville, 1884) Thailand: Chiang Mai 18.80N 98.92E SCAU He495 KY049956 KY049976
Hyarotis quinquepunctatus Fan & Chiba, 2008 China: Hainan 19.03N 109.54E SCAU He114 KY049977
Zographetus satwa (deNicéville, 1884) China: Guangdong 24.88N 113.03E SCAU He442 KY049957 KY049978

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 template DNA, 2 µl 10× buffer, 1.6 µl dNTPs (containing 2.5 mM of each dNTP), 0.8 µl of each primer (10 uM), 0.2 µl Taq Polymerase (2 U/µl), and 13.6 µl ddH2O. The PCR Products were amplified using initial denaturation at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 47 °C (COI) for 45 s, 55 °C (EF-1α) for 1 min, elongation at 72 °C for 1.5 min, and final elongation at 72 °C for 5 min.

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 Table 1).

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 reversible-jump 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.

Results

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.

The pairwise P2K distances among the sequences were variable between genes. The ranges of sequence divergences for two loci and ingroup taxa are: COI (0–12.4%), EF-1α (0–5.0%). For COI, sequence divergence between conspecific individuals ranged from 0 to 0.6%; inter-specific genetic distances ranged from 3.6% to 12.4% with divergences among species averaging 7.9% (Table 2).

Uncorrected pairwise genetic distances (Kimura 2-parameter) for the COI sequences of the genus Scobura species.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 S. cephaloides 102
2 S. cephaloides 553 0.003
3 S. coniata 73 0.096 0.098
4 S. coniata 472 0.096 0.098 0.000
5 S. hainana 471 0.084 0.085 0.059 0.059
6 S. hainana 487 0.084 0.085 0.059 0.059 0.000
7 S. hainana 488 0.084 0.085 0.061 0.061 0.002 0.002
8 S. isota 468 0.115 0.117 0.122 0.122 0.099 0.099 0.099
9 S. isota 538 0.113 0.115 0.118 0.118 0.096 0.096 0.096 0.006
10 S. lyso 465 0.087 0.089 0.061 0.061 0.039 0.039 0.039 0.105 0.101
11 S. lyso 475 0.085 0.087 0.061 0.061 0.036 0.036 0.036 0.101 0.098 0.003
12 S. masutaroi 300 0.092 0.096 0.059 0.059 0.056 0.056 0.056 0.107 0.103 0.052 0.052
13 S. masutaroi 301 0.092 0.096 0.059 0.059 0.056 0.056 0.056 0.107 0.103 0.052 0.052 0.000
14 S. masutaroi 303 0.092 0.096 0.059 0.059 0.056 0.056 0.056 0.109 0.105 0.052 0.052 0.002 0.002
15 S. parawoolletti 116 0.094 0.097 0.089 0.089 0.087 0.087 0.087 0.112 0.108 0.084 0.084 0.084 0.084 0.084
16 S. stellata 36 0.101 0.104 0.104 0.104 0.101 0.101 0.099 0.108 0.105 0.099 0.099 0.092 0.092 0.094 0.070
17 S. woolletti 535 0.094 0.097 0.096 0.096 0.085 0.085 0.085 0.124 0.121 0.092 0.092 0.099 0.099 0.099 0.039 0.074
18 S. woolletti 536 0.092 0.096 0.094 0.094 0.084 0.084 0.084 0.123 0.119 0.090 0.090 0.097 0.097 0.098 0.038 0.072 0.002
19 S. woolletti 537 0.094 0.097 0.096 0.096 0.085 0.085 0.085 0.124 0.121 0.092 0.092 0.099 0.099 0.099 0.039 0.074 0.000 0.002

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.

Figure 1.

Majority-rule consensus tree from the Bayesian analysis (BI) of the concatenated COI and EF-1α sequences. Values at nodes represent the bootstrap support (BS) values of the maximum likelihood (ML) and the posterior probabilities (PP) of BI analyses, respectively (BP/PP).

Clade B is comprised by S. masutaroi and the representatives of S. coniata group (Devyatkin 2004): S. coniata, S. lyso and S. hainana, and the latter two are sister species with strong support (BP = 93, PP = 1.00). The monophyly of S. coniata group including S. masutaroi is strongly supported (BP = 100, PP = 1.00).

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 CuA1 and CuA2 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%.

Figure 2.

Male band of scent scales in the S. coniata group species.

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.

3 Forewing upper side without spots in spaces M3 or M1 and M2 4
Forewing upper side with spots in spaces M1, M2 and M3 6
4 Forewing upper side without spots in spaces M1 and M3, hindwing under side: basal half yellow, distally ferruginous, with five small spots S. cephaloides
Forewing upper side without spot in space M3 5
5 Hindwing under side with a conspicuous rectangular white spot in space CuA2 S. cephala
Hindwing under side without a conspicuous rectangular white spot in space CuA2 S. isota
6 Hindwing upper side without spot in space CuA1, under side with small white spots in spaces Sc+R1, M1-2, M3 and cell S. eximia
Hindwing upper side with the spot in space CuA1 7
7 Forewing cell spots conjoined, subequal 8
Forewing cell spots separated, if conjoined, the lower spot much larger 9
8 Hindwing upper side hyaline spots white S. evansi
Hindwing upper side hyaline spots yellow S. masutaroi
9 Forewing upper side the spot in space CuA2 triangular, and with a linear stigma crossing the spots in spaces CuA1 and CuA2 S. coniata
Forewing upper side the spot in space CuA2 not as above 10
10 Forewing upper side the spot in space CuA1 narrow, hindwing upper side without spot in space S. lyso
Forewing upper side the spot in space CuA1 broad 11
11 Hindwing upper side spot in space M3 tiny dot, forewing upper side cell spots cell spots conjoined S. hainana
Hindwing upper side spot in space M3 significant, forewing upper side cell spots cell spots separated S. phuongi

Scobura masutaroi Sugiyama, 1996

Fig. 3

Scobura masutaroi Sugiyama, 1996: 9 (Type locality: Dujiangyan, Sichuan, China)

Scobura mouchai Krajcik, 2013: 2, syn. n. (Type locality: Fengxian, Shaanxi, China)

Material examined

1♂, 1♀, Nibashan, Rongjing, Sichuan, 26.VII.2009, Min Wang; 1♂, Jialingjiang, Fengxian, Shaanxi, 15.VII.2010, Min Wang.

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 CuA2 yellow; hindwing with spots in spaces CuA1 and M1-M2 yellow. Wing under side: forewing costal and submarginal spots yellow; hindwing all veins and submarginal spots from spaces Sc+R1 to CuA2 yellow; and all yellow submarginal spots conjoined both forewing and hindwing.

Figure 3.

Scobura masutaroi Sugiyama, 1996 (Sichuan): A, B male C, D female; scale bar 10 mm.

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.

Figure 4.

Male genitalia of Scobura masutaroi Sugiyama, 1996. (Sichuan). A Genitalia ring, lateral view; B aedeagus and juxta. C valva, inner view; D tegument, dorsal view.

Female genitalia (Fig. 5): Papillae anales rectangular, covered with setae; anterior lamella U-shaped with sclerotization; posterior lamella triangular with upper margin arched; ductus bursae membranous and short; copulatrix bursa elongate, membranous.

Figure 5.

Female genitalia of Scobura masutaroi Sugiyama, 1996 (Sichuan)

Distribution

China (Sichuan, Shaanxi).

Acknowledgements

We are grateful to Drs Liu-Sheng Chen (Shihezi University, Xinjiang, China), Hou-shuai Wang and Hai-ming Xu (SCAU) for collecting the specimens. Materials of some species were provided by the Kyushu University museum, Dr Osamu Yata and Mr Hiroaki Onodera. The work is supported by the Natural Science Foundation of China (NSFC- 31172136 and -31471984).

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