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

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 Elwes & Edwards, 1897 was recently revised by Fan et al. (2010), who recognized 14 species. The genus , however, includes another species, , 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, , 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 , based on molecular evidence. By comparing molecular and morphological evidence, we examine whether is a synonym of .

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 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 , except for some specimens in and Mr. Hiroaki Onodera’s private collection.

Table 1.

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

Insect Collection, Department of Entomology, South China Agriculture University

Kyushu University museum

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 PageBreakPageBreaktemplate 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 and 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 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 and ML were used to evaluate branch support.

Bayesian inference

Markov Chain Monte Carlo

bootstrap values

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).

Table 2.

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

		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
1	Scobura cephaloides 102
2	Scobura cephaloides 553	0.003
3	Scobura coniata 73	0.096	0.098
4	Scobura coniata 472	0.096	0.098	0.000
5	Scobura hainana 471	0.084	0.085	0.059	0.059
6	Scobura hainana 487	0.084	0.085	0.059	0.059	0.000
7	Scobura hainana 488	0.084	0.085	0.061	0.061	0.002	0.002
8	Scobura isota 468	0.115	0.117	0.122	0.122	0.099	0.099	0.099
9	Scobura isota 538	0.113	0.115	0.118	0.118	0.096	0.096	0.096	0.006
10	Scobura lyso 465	0.087	0.089	0.061	0.061	0.039	0.039	0.039	0.105	0.101
11	Scobura lyso 475	0.085	0.087	0.061	0.061	0.036	0.036	0.036	0.101	0.098	0.003
12	Scobura 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	Scobura 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	Scobura 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	Scobura 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	Scobura 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	Scobura 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	Scobura 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	Scobura 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

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 is weakly supported (BP = 44, PP = 0.87). Within the genus, although support for the basal clades was low, the species included here are clearly distinguished from each other, and formed four clades: the clade (which only included two representative specimens), Clade A, the clade (only with two representative specimens), and Clade B. Clade A is comprised by + ( + ) and receive high bootstrap support and posterior probability (BP = 99, PP = 1.00). We hereafter called the clade group.

Figure 1.

Majority-rule consensus tree from the of the concatenated COI and EF-1α sequences. Values at nodes represent the values of the and the of BI analyses, respectively (BP/PP).

Bayesian analysis

bootstrap support

maximum likelihood

posterior probabilities

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

In all the analyses, is sister to Clade B, with moderate support (BP = 63, PP = 0.92), whereas the relationships between and the other clades (Clade A, and Clade B) remain unresolved.

Discussion

Although our phylogenetic analyses do not strongly support the monophyly of the genus , two strongly supported monophyletic groups within the genus are recognized: the group and the group. The members of the 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. is nested within this group. In our present analyses, two individuals (He 300, 301) of from Nibashan, Sichuan (close to Dujiangyan, Sichuan, the type locality of ) and an individual (He303) from Jialingjiang, Fengxian, Shaanxi (the type locality of ) 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 group range from 3.3% to 6.1% with divergences between species averaging 4.5%, while divergence between individuals of from Sichuan and Shaanxi province was 0.2%.

Figure 2.

Male band of scent scales in the 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 PageBreakis identical to 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, and , which are not included in the present study, likely also belong to this group.

A well-support clade comprised by , and 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 Riley, 1923 was proposed with 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 . Based on morphological characters, the group also includes (Evans, 1914).

Taxonomic account

The key given by Fan et al. (2010) is modified to include . The couplets leading to only are included here. Couplets beyond 11 in the original increase their number by one.

Sugiyama, 1996

Fig. 3

Figure 3.

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

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

Krajcik, 2013: 2,

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 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.

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 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 Sugiyama, 1996 (Sichuan)

Distribution.

China (Sichuan, Shaanxi).

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	Scobura cephaloides
–	Forewing upper side without spot in space M3	5
5	Hindwing under side with a conspicuous rectangular white spot in space CuA2	Scobura cephala
–	Hindwing under side without a conspicuous rectangular white spot in space CuA2	Scobura 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	Scobura 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	Scobura evansi
–	Hindwing upper side hyaline spots yellow	Scobura masutaroi
9	Forewing upper side the spot in space CuA2 triangular, and with a linear stigma crossing the spots in spaces CuA1 and CuA2	Scobura 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	Scobura 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	Scobura hainana
–	Hindwing upper side spot in space M3 significant, forewing upper side cell spots cell spots separated	Scobura phuongi