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Molecular and morphological evidence reveals hidden new taxa in Ochlodes ochraceus (Bremer, 1861) (Lepidoptera, Hesperiidae, Hesperiinae) from China
expand article infoLijuan Zhu, Yongxiang Hou, Hideyuki Chiba§, Yohei Osada|, Zhenfu Huang, Sergey Yu. Sinev#, Min Wang, Xiaoling Fan
‡ South China Agricultural University, Guangzhou, China
§ B. P. Bishop Museum, Honolulu, United States of America
| Osaka Museum of Natural History, Osaka, Japan
¶ Southwest University of Science and Technology, Mianyang, China
# Zoological Institute of the Russian Academy of Sciences, Saint-Petersburg, Russia
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Abstract

Two new species of Ochlodes Scudder, 1872, Ochlodes pseudochraceus Zhu, Fan & Wang, sp. nov. and Ochlodes cryptochraceus Zhu, Fan & Chiba, sp. nov., are found in China and described, and Ochlodes rikuchina (Butler, 1878) stat. rev. is restored. A lectotype is designated for Pamphila ochracea Bremer, 1861, and a neotype is designated for Pamphila rikuchina Butler, 1878. Overall, the two new species are similar to Ochlodes ochraceus (Bremer, 1861). They, however, can be distinguished from the latter and other species in the genus: O. pseudochraceus has long radial spots in spaces R3-5, and the lateral process of the phallus gradually widens at the distal half in male genitalia; O. cryptochraceus has the lateral process of the phallus enlarged only at the distal tip. Based on the phylogenetic analyses of the mitochondrial COI gene, members of currently defined O. ochraceus are grouped into four clades. The genetic distances between O. pseudochraceus and O. ochraceus, O. cryptochraceus and O. ochraceus, O. rikuchina and O. ochraceus, and O. pseudochraceus and O. cryptochraceus are 3.2%, 2.1%, 1.9%, and 2.7%, respectively. Based on the molecular and morphological evidence, O. pseudochraceus, O. cryptochraceus, and O. rikuchina are treated to be distinct species. The adult habitus and male and female genitalia of the new species are illustrated as well as those of O. ochraceus and O. rikuchina.

Key words

Genitalia, Hubei, new species, phylogeny, Shaanxi, Sichuan, taxonomy, Zhejiang

Introduction

The genus Ochlodes Scudder, 1872, described with Hesperia nemorum Boisduval, 1852 as the type species, belongs to the family Hesperiidae and is distributed in the Oriental, Palearctic, and Nearctic regions. Evans (1949, 1955) recognized 16 species and 23 subspecies within the genus. Chiba and Tsukiyama (1996) revised the genus based on wing pattern, male genitalia, and geographical distribution. A total of 23 species and 17 subspecies were recognized and classified into the following four groups: venata complex, subhyalina group, bouddha group, and a miscellaneous group. Among these, five species are distributed in the Nearctic region and the remainder in the Palearctic and Oriental regions.

China is the most species-rich area for the genus Ochlodes, with 16 species recorded to date (Burns 1992; Chiba and Tsukiyama 1996; Cao et al. 2019; Hsu et al. 2006; Hsu et al. 2019). During our revisional study of the genus, we observed that 16 specimens of O. ochraceus collected in China (Shaanxi, Sichuan, Zhejiang, and Hubei), Japan, and Russia (type locality) were grouped into four clades based on the COI gene, corresponding to two samples from Russia, six samples from Japan, three samples from Zhejiang, China, and five samples from Shaanxi, Sichuan, and Hubei, China. Based on careful examination, we found that the specimens of the four clades differed in genitalic morphology, and those from Zhejiang and other localities in China differed from all other species in the genus. Therefore, four distinct species can be recognized, and the specimens from Shaanxi, Sichuan, Hubei, and Zhejiang in China represent two new species.

Materials and methods

Morphological study

The specimens examined in this study were collected using an insect net and deposited at the South China Agricultural University (SCAU), Guangzhou, China. The specimens in the following institutional and private collections were also examined: Northeast Forestry University (NEFU), Harbin, China; private collection of Hideyuki Chiba (HC), Fukuoka, Japan; Osaka Museum of Natural History (OMNH), Osaka, Japan; Hokkaido University Museum (HUM), Hokkaido, Japan; Leibniz Institute for the Analysis of Biodiversity Change, Zoological Research Museum Alexander Koenig (Zoologisches Forschungsmuseum Alexander Koenig, ZFMK), Bonn, Germany; Zoological Institute of Russian Academy of Sciences (ZIN), Saint Petersburg, Russia; Natural History Museum, London, United Kingdom (NHMUK, formerly BMNH). Images of the type of O. rikuchina were used with permission (copyright: Trustees Natural History Museum, photograph R. Crowther). All adult photographs were captured using a SONY DSC-RX100 camera. The abdomens were removed and macerated in 10% NaOH solution to examine the male and female genitalia. Genitalia were photographed using the Keyence VHX-5000 ultra-depth of field 3D microsystem. The wing venation was examined according to the method Hou et al. (2021) outlined and photographed using a smartphone. All photographs were processed by Abode Photoshop CC and Abode Illustrator CC 2018. The terminology for adults and genitalia follows Chiba and Tsukiyama (1996) and Fan et al. (2010).

Molecular analysis

Based on the classification of Chiba and Tsukiyama (1996), 29 specimens of Ochlodes were sampled as ingroups, representing two species placed in the miscellaneous group: Ochlodes linga Evans, 1939 and O. ochraceus, and five species placed in all other species groups. Whenever possible, samples from the type localities or near the type localities were included for previously described taxa. The COI barcodes of all 29 specimens were sequenced, and the sequences were deposited in GenBank. The sequence information of two species of Hesperia (H. meskei and H. attalus) was downloaded from GenBank (https://www.ncbi.nlm.nih.gov) as outgroups based on prior information (Yuan et al. 2015b). Detailed information on materials and accession numbers is provided in Table 1. Our previous studies referred to details of the DNA extraction, amplification, and sequencing protocols (Huang et al. 2019a; Hou et al. 2021). Genetic distances were calculated using Kimura 2-parameter models in MEGA 7.0 (Kumar et al. 2016). Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) methods. ML analyses were performed using IQ-TREE 2.2.1 (Minh et al. 2020) on a local computer. The data were partitioned into codon positions and models (1st: HKY+F+G4, 2nd: TN+F+I, and 3rd: F81+F+I) were selected using ModelFinder (Kalyaanamoorthy et al. 2017) in IQ-TREE 2.2.1 (Minh et al. 2020). The nucleotide substitution models were estimated under the Bayesian Information Criterion (BIC) with FreeRate heterogeneity, which relaxes the assumption of gamma-distributed rates. Both Ultrafast bootstrap (UFBoot) (Minh et al. 2013) and SH-aLRT branch test (Guindon et al. 2010) were performed with 1000 replicates to evaluate branch support, and the tree with the highest likelihood was selected. The BI analyses were performed using MrBayes v. 3.2.6 on CIPRES Science Gateway 3.3 (http://www.phylo.org/) (Miller et al. 2010) with Markov Chain Monte Carlo (MCMC) randomization in MrBayes using XSEDE 3.2.6 (Ronquist et al. 2012). Two independent runs were performed, and the starting tree was set to a random tree. Four Markov chains (three hot chains and one cold chain) ran 5 × 106 generations simultaneously, sampling every 1000 generations, with the first 25% of sampled trees discarded as burn-in. Tracer v. 1.7.2 (Rambaut et al. 2018) was used to determine the standard deviation of the split frequency value, which was < 0.01, and the effective sample size (ESS) > 200, indicating that the runs reached stationarity. Bayesian posterior probabilities (PP) were used to evaluate branch support. Trees were visualized using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Table 1.

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

Taxon Data Locality Voucher number Accession number
Ochlodes venatus VIII. 2009 Beijing, China SCAU_He2718 OQ452925
Ochlodes venatus VII.2018 Heilongjiang, China SCAU_He2602 OQ452926
Ochlodes similis VI. 2021 Songpan, Sichuan, China SCAU_He2650 OQ452930
Ochlodes similis VI. 2010 Beijing, China SCAU_He2658 OQ452929
Ochlodes sagittus VII.2018 Kangding, Sichuan, China SCAU_He2613 OQ452932
Ochlodes sagittus VI. 2021 Songpan, Sichuan, China SCAU_He2647 OQ452931
Ochlodes bouddha VI. 2019 Yaan, Sichuan, China SCAU_He2620 OQ452947
Ochlodes bouddha VII.2019 Ankang, Shaanxi, China SCAU_He2685 OQ452946
Ochlodes subhyalinus VIII. 2018 Hanzhong, Shaanxi, China SCAU_He2646 OQ452927
Ochlodes subhyalinus VI. 2017 Tianshui, Gansu, China SCAU_He2617 OQ452928
Ochlodes linga V. 2018 Hanzhong, Shaanxi, China SCAU_He2696 OQ452941
Ochlodes linga VI. 2018 Baoji, Shaanxi, China SCAU_He2697 OQ452940
Ochlodes linga V. 2018 Baoji, Shaanxi, China SCAU_He2698 OQ452939
Ochlodes ochraceus VI. 2019 Shennongjia, Hubei, China SCAU_He2605 OQ452945
Ochlodes ochraceus VI. 2018 Yulin, Shaanxi, China SCAU_He2632 OQ452944
Ochlodes ochraceus VI. 2019 Shennongjia, Hubei, China SCAU_He2677 OQ452943
Ochlodes ochraceus VI. 2021 Baoji, Shaanxi, China SCAU_He2678 OQ452942
Ochlodes ochraceus VII. 2022 Mianyang, Sichuan, China SCAU_He2746 OQ749886
Ochlodes ochraceus VIII. 2016 Ningbo, Zhejiang, China SCAU_He2614 OQ452935
Ochlodes ochraceus VIII. 2016 Ningbo, Zhejiang, China SCAU_He2637 OQ452934
Ochlodes ochraceus VIII. 2016 Ningbo, Zhejiang, China SCAU_He2676 OQ452924
Ochlodes ochraceus VII.2022 Ueda-shi, Japan SCAU_He2729 OQ452937
Ochlodes ochraceus VII.2021 Ueda-shi, Japan SCAU_He2730 OQ452933
Ochlodes ochraceus VII. 2017 Japan SCAU_He2734 OQ749884
Ochlodes ochraceus VII. 2017 Japan SCAU_He2736 OQ749885
Ochlodes ochraceus VII. 2021 Iwate-ken, Japan SCAU_He2726 OR058650
Ochlodes ochraceus V. 1990 Aichi-ken, Japan SCAU_He2727 OR058651
Ochlodes ochraceus III.2018 Primorsky Kray, Russia SCAU_He2728 OQ452938
Ochlodes ochraceus VII.2015 Primorsky Kray, Russia SCAU_He2732 OQ452936
Hesperia meskei X.1997 Florida, United States CSU-CPG-LEP001666 GU685651
Hesperia attalus III.2002 California, United States AAE3790 GU685041

Combining DNA sequence data with other kinds of characters produces a more precise taxonomic framework (DeSalle et al. 2005). DNA barcoding helps recognize cryptic species (Nolasco and Valdez-Mondragón 2022). In this study, three different criteria were adopted, namely, morphological characters, monophyly, and genetic distance, for species delimitation based on the unified species concept described by de Queiroz (2005). If two taxa are recovered as monophyletic and have consistent morphological differences, and the genetic distance between them is not less than 0.8% (the genetic distance between Ochlodes similis and O. sagittus, which are morphologically two clearly distinct species with sympatric distribution), they are treated as two distinct species.

Results and discussion

Phylogenetic analysis

The phylogenetic tree (Fig. 1) constructed using the COI barcoding region shows that the members of the genus Ochlodes are clustered together with strong support (PP/UFBoot = 1/100). The following five lineages are recognized: 1) venatus complex, including O. venatus, O. similis, and O. sagittus; 2) O. bouddha; 3) O. linga; 4) O. subhyalinus; and 5) O. ochraceus. The miscellaneous group of Chiba and Tsukiyama (1996) is divided into two distant clades. The 16 samples of O. ochraceus are clustered together and recovered in four subclades: O. ochraceus from Primorsky Kray, Russia; clade A from Japan; clade B from Zhejiang, China; and clade C from Shaanxi, Sichuan, and Hubei in China. The genetic distance range among species calculated with COI barcode is 0.8–9.9%, of which the distance range among the three species within venatus complex is 0.8–1.6%, the distance among the four subclades of O. ochraceus is 1.9% between O. ochraceus and clade A, 3.2% between clade A and clade B, 2.1% between clade A and clade C, 3.2% between O. ochraceus and clade B, 2.1% between O. ochraceus and clade C, and 2.7% between clade B and clade C (Table 2). The traditional O. ochraceus clade, characterized by a wider and darker wing margin, is named the ochraceus complex.

Figure 1. 

Phylogenetic tree of Ochlodes based on COI barcode region, using the ML and BI methods. Values at nodes represent the posterior probabilities (PP) of BI analyses and Ultrafast bootstrap values (UFBoot) of the ML analyses.

Table 2.

Genetic distances among Ochlodes species based on COI barcodes.

1 2 3 4 5 6 7 8 9
O. venatus
O. similis 0.015
O. sagittus 0.016 0.008
O. bouddha 0.068 0.070 0.072
O. linga 0.066 0.064 0.067 0.065
O. subhyalinus 0.059 0.058 0.061 0.071 0.059
O. ochraceus 0.069 0.070 0.073 0.086 0.073 0.067
O. rikuchina 0.080 0.082 0.085 0.099 0.082 0.076 0.019
O. pseudochraceus 0.076 0.078 0.081 0.084 0.079 0.070 0.032 0.032
O. cryptochraceus 0.075 0.076 0.080 0.086 0.076 0.071 0.021 0.021 0.027

Taxonomy of the ochraceus complex and relatives

We examined the syntype of O. ochraceus from Primorsky Kray, Russia (https://www.zin.ru/collections/Lepidoptera), deposited in ZIN (Fig. 3A). Despite the label indicating its status as a lectotype, such a designation has not been published. Therefore, we herein designate the male specimen as the lectotype. Apart from the lectotype, 12 other specimens of O. ochraceus (five from Primorsky Kray, two from Amur, Russia, and five from Heilongjiang, China) were examined. We also examined the specimen whose labels indicate “Type, the locality Miyanoshita” at NHMUK (Fig. 3D) and 55 specimens of O. rikuchina from Japan. All the taxa in the ochraceus complex share consistent and distinct morphological characters (Table 3). Ochlodes ochraceus from Russia and O. rikuchina (clade A) from Japan share the following characters in male genitalia: the dorsodistal process is finger-like, the ventrodistal process of the valva is broad and round distally, and the lateral process of the phallus is not enlarged. In O. ochraceus, however, the tegumen extends distally, and the uncus is wide. In contrast, in O. rikuchina, the tegumen does not extend distally, and the uncus is narrow. Members of clade B can be distinguished from the other taxa by their wing patterns and male genitalia. The spots in spaces R3–R5 on the forewing upper side are short and radial and away from the discocellular vein, and the lateral long process of the phallus is gradually widened with a row of small spines along the dorsal margin. In the other taxa of the complex, these spots are long and reach the discocellular vein, and the process of the phallus is only enlarged at the distal tip (clade C) or not significantly widened (clade A). In addition, the stigma of these taxa is divided into three parts: the first part in space CuA1 and the second in space CuA2 are markedly different, whereas the third part in space CuA2 is vague. The first and the second parts of O. ochraceus are crescent- and spindle-shaped, respectively, differing from those in the other taxa (Fig. 2). Therefore, we believe that the currently recognized O. ochraceus is not a single species but includes hidden species. Based on morphological characters and molecular evidence, we describe clades B and C as two new species below: O. pseudochraceus Zhu, Fan & Wang, sp. nov. and O. cryptochraceus Zhu, Fan & Chiba, sp. nov. Additionally, clade A is recognized as a valid species, and O. rikuchina stat. rev. is restored.

Figure 2. 

Wing venation and stigma of ochraceus complex. A O. pseudochraceus sp. nov., holotype, Zhejiang, SCAU He2614 B O. cryptochraceus sp. nov., holotype, Hubei, SCAU He2605 C O. rikuchina stat. rev., neotype, Iwate, SCAU He2726 D O. ochraceus, Russia, SCAU He2728. Scale bar: 0.5 cm.

Figure 3. 

Adults of four Ochlodes species A–C O. ochracea A lectotype, male, Primorsky Kray, Russia B male, Primorsky Kray, Russia, SCAU He2728 C male, Amur, Russia D–G O. rikuchina stat. rev. D female, Miyanoshita, Japan (NHMUK) E neotype, male, Iwate, Japan (designated herein), SCAU He2726 F male, Japan, SCAU He2736 G female, Japan, SCAU He2727 H Augiades ochracea var. ampittiformis. holotype, female, Tokyo, Japan I–K O. pseudochraceus sp. nov. I holotype, male, Zhejiang, SCAU He2614 J paratype, male, Zhejiang, SCAU He2676 K paratypes, female, Zhejiang, SCAU He2637 L–N O. cryptochraceus sp. nov. L holotype, male, Hubei, SCAU He2605 M paratype, male, Shaanxi, SCAU He2678 N paratypes, female, Shaanxi, SCAU He2632.

Table 3.

Comparison of morphological differences among four species of ochraceus complex in Ochlodes.

pseudochraceus O. cryptochraceus rikuchina O. ochraceus
Color of wing margin dark dark pale brown pale brown
Stigma thin and long, not aligned at CuA2 vein. thick and long, not aligned at CuA2 vein. thick and short, aligned at CuA2 vein. thick and short, aligned at CuA2 vein.
Saccus thin and long thin and long thin and short thick and short
Phallus lateral process gradually enlarged at distal half and serrated, not reaching the tip of phallus lateral process only enlarged at the distal tip lateral process not enlarged, distal half with serrated reaching the tip of phallus lateral process not enlarged, distal half with serrated reaching the tip of phallus
Uncus narrow slightly narrow narrow wide
Tegumen not extend distally slightly extent distally not extent distally extend distally

In the previous studies, O. ochraceus has been recorded in Zhejiang, China (Tong 1993; Chou 1994; Yang et al. 1994; Chu et al. 2017). However, the specimens illustrated by Tong (1993), Chou (1994) (female), and Yang et al. (1994) are O. linga. We observed no specimens or photographs of true O. ochraceus collected in Zhejiang. Similarly, the specimens illustrated in most previous studies (Chiba and Tsukiyama 1996: pl. 1 figs 20, 22; Cai et al. 2011; Yuan et al. 2015a; Wu and Hsu 2017) are Ochlodes cryptochraceus Zhu, Fan & Chiba, sp. nov., whereas the specimen illustrated in Chu et al. (2017) is Ochlodes pseudochraceus Zhu, Fan & Wang, sp. nov.

Butler (1878) described Pamphila rikuchina based on an unstated number of specimens from Rikuchu, an old name of northeastern Japan which includes most of current Iwate and a part of Akita prefecture, erroneously naming it after “Rikuchin” from the handwriting of M. A. Fenton (Matsuda 1995). In addition, Butler (1878) did not illustrate this species, nor specify the sex of the specimen(s) he examined. However, his description, “primaries with two ochreous spots at the end of the cell (the upper one punctiform), secondaries with an arched series of five ochreous spots on the discal” is clear enough to recognize the type is of female. Evans (1949) mentioned a female type specimen from Japan. We examined the female specimen of Pamphila rikuchina deposited in NHMUK (Fig. 3D), which indicates that the female specimen collected from Miyanoshita in [18]87 is not the syntype examined by Butler (1878). Blanca Huertas (pers. comm.) conducted a thorough search at NHMUK, including the Evans’ reference collection, but she did not find any other specimen labelled as the type of this taxa or with Butler’s label, implying that the syntype(s) is likely lost. Considering this, a neotype designated for this name rikuchina is necessary to stabilize the taxon.

According to Article 75.3 of ICZN (1999), the exceptional need for this neotype designation, apart from the loss of the name-bearing syntype specimen(s), was based on the following: (1) The status of O. rikuchina (Butler, 1878) has not been settled, and it was treated as a synonym of O. ochraceus (Evans 1949; Chiba and Tsukiyama 1996; Yuan et al. 2015a) or as a subspecies of O. ochraceus (Kudrna 1974; Kawazoé and Wakabayashi 1976; Lee 1982). Our morphological and molecular studies show that O. rikuchina is a valid species. (2) This species can be distinguished from the other taxa in the ochraceus complex by the club of antenna being thin and long, the male genitalia having the tegumen that does not extend distally, the uncus being narrow, and the phallus with distal half of lateral process not enlarged. (3) The neotype should be a female specimen based on the origin description (Butler 1878), but it is difficult to identify species based on a female in Hesperiidae, given that most specimens in the genus Ochlodes are males. To secure the nomenclatural stability, we designated a male specimen from Iwate (type locality) as a neotype for O. rikuchina based on our morphological and molecular studies.

Neotype designation: Omorisawa, Isawa, Oshu-shi, Iwate prefecture, Japan, 31.VII.2010, S. Sakuratani leg// SCAU_He 2726// (SCAU) (Fig. 3E). For detailed description, see Taxonomy below.

Matsumura (1919) described Augiades ochracea var. ampittiformis based on a single female specimen from Nakano, near Tokyo, Japan, which is currently deposited in HUM. According to Article 73.1.2 of ICZN (1999), we consider that this female specimen to be the holotype fixed by monotypy based on the statement of only ‘one female specimen’ provided in the original description. We examined the holotype of Augiades ochracea var. ampittiformis (Fig. 3H) and considered that the characters of the original description, “both wings with much smaller spots, an indistinct tiny anterior spot on the discocellular, and two tiny spots respectively in the 4th and 5th interspaces”, represent only an individual variation of O. rikuchina. This was due to the size of the wing pattern of O. rikuchina being slightly variable among individuals. The upper spot in the discal cell is indistinct (Fig. 3H; Chiba and Tsukiyama 1996: pl 3 fig. 1) or ranges from a small dot (Kawazoé and Wakabayashi 1976) to a spot slightly smaller than the lower spot in the female (Fig. 3G); In contrast, in the other species of the ochraceus complex, the upper spot is not smaller than the lower spot. Therefore, we treat ampittiformis as a junior subjective synonym of rikuchina.

Lee (1982) noted that it seemed reasonable to regard the Korean population as the nominate subspecies ochraceus. After carefully examining the photographs, it is tentatively concluded that those illustrated in Lee (1982: pl. 63 fig. 243 excluding C, D) are of O. ochraceus. Further investigation, however, is required.

Chiba and Tsukiyama (1996) treated three subspecies of O. venatus sensu Evans (1949), similis, sagittus, and hyrcana (now sylvanus), as distinct species and placed them in the venata complex. We follow the treatment based on morphological characters and their sympatric distribution.

Morphological characters are considered inadequate for the identification of skipper butterflies (Ackery et al. 1999), and it is common, particularly in Hesperiidae, for a complex of sibling species with similar morphological characters to be recognized as one species (Hebert et al. 2004; Burns and Janzen 2005; Huang et al. 2019b; Huang 2021). As taxonomic and biological research progresses, the relationships among species become clearer, often resulting in the recognition of hidden taxa. Further study is required to investigate the possible sympatry of the new species with O. ochraceus in Zhejiang.

Taxonomic accounts

Ochlodes ochraceus (Bremer, 1861)

Figs 3A–C, 4G, H

Pamphila ochracea Bremer, 1861: 473 (type locality, original label: Ussuri). Lectotype (location: Primorsky Kray, Russia), designated herein.

Ochlodes ochracea: Evans 1949: 353; Chiba and Tsukiyama 1996: 16, pl 1 fig. 19; Tuzov et al. 1997: 129; Wang 1999: 286; Zhou and Zhu 2003: 196.

Ochlodes ochracea rikuchina: Lee 1982, pl 63 figs 243A, B.

Diagnosis

Antenna with black and white stripes, club thick. Male genitalia (n = 5): tegumen extend distally; uncus wide; valva with dorsodistal process finger-like and round at tip, ventrodistal process widen and round; phallus with lateral process longer than subzonal sheath, distal half not enlarged and serrated reaching tip of phallus.

Figure 4. 

Male genitalia of four Ochlodes species A, B O. pseudochraceus sp. nov. A holotype, male, SCAU He2614 B paratypes, male, SCAU He2676 C, D O. cryptochraceus sp. nov. C holotype, male, SCAU He2605 D paratypes, male, SCAU He2678 E, F O. rikuchina stat. rev. E male, SCAU He2726 F male, SCAU He2736 G, H O. ochraceus G male, SCAU He2728 H male, Amur, Russia.

Specimens examined

Lectotype , ♂, Primorsky Kray, Russia (ZIN); 1♂, SCAU_He2728, 2.III.2018, Primorshy kray, Chuguevsky district, Russia (OMNH); 1♂, SCAU_He2731, 7.VII.2015, Primorshy kray, Spassky district, Russia (OMNH); 1♂, SCAU_He2732, 7.VII.2015, Primorsky Kray, Spassky district, Russia (OMNH); 1♂, 7.III.2018, Primorsky Kray, Chuguevsky district, Russia, Golovizin V. Col; 1♂, 7 VII 2016, Primorsky Kray, Chuguevsky district, Russia, Golovizin V. Col; 2♂, 6.VII.1974, Maoershan, Heilongjiang Province (NEFU); 1♂, Amur (ZFMK Lep153522); 1♂ VI 1927, Maoershan, Heilongjiang Province (ZFMK); 1♂, 7.VII.2016, Acheng district, Heilongjiang Province, (HC).

Distribution

China (Heilongjiang, Jilin); Russia (Far East); Korea.

Ochlodes rikuchina (Butler, 1878), stat. rev.

Figs 3D–H, 4E, F, 5C

Pamphila rikuchina Butler, 1878: 285. Neotype ♂, designated herein (type locality: Oshu-shi, Iwate prefecture, Japan).

Augiades ochracea var. ampittiformis Matsumura, 1919: 737–738 (type locality: Nakano near Tokyo, Japan).

Ochlodes ochracea rikuchina: Kudrna 1974; Kawazoé and Wakabayashi 1976.

Specimens examined

Neotype , ♂, SCAU_He 2726, 31.VII.2010, Omorisawa, Isawa, Oshu-shi, Iwate prefecture, Japan, S. Sakuratani leg; type, ♀, Miyanoshita, Japan (NHMUK//BMNH(E) #1055523); 1♂, SCAU_He 2729, 1♂, SCAU_He 2730, 12.VII.2021, Ueda-shi, Japan; 1♂, SCAU_He 2734, Japan; 1♀, SCAU_He2727, 17.VIII.1990, Aichi-pref., Mt. Naganoyama, Japan, Yamanaka leg.; 1♂, 31.VII.2010, S. Sakuratani leg., (HC); 1♂,7.VII.2001, Miyagi-pref., Ishinomaki, Japan, S. Sakuratani leg., (HC); 8♂, 7♀, 17.VII.1997, Gunma-pref., Mt. Haruna, Japan, H. Chiba leg., (HC); 1♂, 28.VII.1976, Gunma-pref., Hotaka, Japan, H. Chiba leg., (HC); 7♂, 1♀, 5.VI.1977, Tokyo, Itsukaichi, Japan, H. Chiba leg., (HC); 1♂, 30.V.1971, Tokyo, Okutama Japan, (HC); 1♂, 4.VII.1996, Tochigi-pref, Shinobara, Japan, S. Hashimoto leg., (HC); 1♂, 18.VII.1976, Nagano-pref, Lake Matsubara Japan, (HC); 2♂, 5.V.2007, Nagano-pref, Azumi Japan, (HC); 1♂, 7.VII.1996, Nagano-pref., Togakushi, Japan, A. Okubo leg., (HC); 1♂, 15.VII.1995, Nagano-pref., Kaida, Japan, Yamanaka leg., (HC); 3♂, 1♀, 2.VIII.1997, Nagano-pref., Kaida, Japan, Yamanaka leg., (HC); 4♂, 1♀, 17.VIII.1990, Aichi-pref., Mt. Naganoyama, Japan, Yamanaka leg., (HC); 1♂, 1♀, 20.VI.1995, Aichi-pref. Iwanami, Japan, Yamanaka leg., (HC); 1♂, 27.V.1990, Gifu-pref., Nagataki, Japan, H. Yamanaka leg., (HC); 1♂, 16.VI.1991, Okayama-pref., Niimi, Japan, Osaka leg., (HC); 2♂, 28.VI.1998, Okayama-pref., Kawakami Japan, (HC); 1♂, 20.V.1998, Miyazaki-pref., Takachiho, Japan, M. Murakami leg., (HC); 1♂, 12.VIII.1998, Miyazaki-pref., Takachiho, Japan, Murakami leg., (HC).

Diagnosis

Antenna with black and white stripes, club thin and long. Male genitalia: tegumen not extend distally; uncus narrow; phallus with lateral process almost equal to subzonal sheath and not enlarged, distal half serrated.

Redescription

Forewing length 15 ± 0.5 mm in males and 15.5 mm in females (Fig. 3E–G). Antenna longer than half length of forewing. Labial palpi: second segment porrect and covered with long brown hairs, third segment short.

Male (Fig. 3E, F). Forewing upper side: ground color brown with orange spots. Spots in spaces R1–R5 long radial and connected to discocellular vein; spots in space M1 to CuA2 form a broad brand, of which spot in space M1 very small; cell orange; stigma black-grey, thin and short. Hindwing upper side: ground color same as forewing, central part including cell and spaces Rs-CuA2 orange. Wing under side: ground color orange-yellow, with spots orange, and blurred.

Female (Fig. 3G). Different from male in reduced spots in spaces R1, 2; only a couple of cell spots present on forewing; cell spot and spots in spaces Rs-CuA2 short and small on hindwing.

Male genitalia (n = 3) (Fig. 4E, F). Tegumen not extend distally and shorter than uncus; uncus narrow and bifurcated at distal tip, with closely aligned arms; gnathos well-developed and bifid; valva long and narrow, dorsodistal process narrow and long, ventrodistal process widened and angled at the tip; phallus with lateral process almost equal to subzonal sheath, distal half not enlarged and serrated reaching the tip of phallus; juxta horseshoe shaped.

Female genitalia (Fig. 5C). Papilla analis irregularly triangular in dorsal view; lamella antevaginalis trilobed shaped, lamella postvaginalis U-shaped, with upper margin straight and densely covered with fine hairs and spines; ductus bursae thick and short; bursa copulatrix oblong and membranous.

Figure 5. 

Female genitalia of four Ochlodes species A O. pseudochraceus sp. nov., SCAU He2637 B O. cryptochraceus sp. nov., SCAU He2678 C O. rikuchina stat. rev., SCAU He2727.

Distribution

Japan (Honshu, Shikoku and Kyushu).

Ochlodes pseudochraceus Zhu, Fan & Wang, sp. nov.

Figs 3I–K, 4A, B, 5A

Ochlodes ochracea: Chu et al. 2017: 421.

Type material

Holotype : ♂. 1.VIII.2016, Simingshan, Ningbo City, Zhejiang province, P. R, China, leg. Houshuai Wang & Shuqin Ji. SCAU_He2614. Paratypes: 1♂. 1♀. SCAU_He2637 (female) and SCAU_He2676 (male) with the same data as holotype.

Diagnosis

Spots in spaces R3–R5 on forewing upper side radial and far from discocellular vein. Male genitalia: lateral process of phallus with distal half gradually enlarged, with a row of small spines. Female genitalia: upper margin of lamella postvaginalis straight.

Description

Forewing length 15 mm in males and 14 mm in females (Fig. 3I-K). Antenna longer than half length of forewing. Labial palpi, second segment porrect and covered with long brown hairs, third segment short.

Male (Fig. 3I, J). Forewing upper side: ground color dark brown with orange-yellow spots. Spots in spaces R3–R5 short and radial, far from discocellular vein; spots in space M1 to CuA2 form a broad brand, of which spot in space M1 very small; cell orange-yellow; stigma black-grey, thin and long. Hindwing upper side: ground color same as forewing, central part including cell and spaces Rs-CuA2 orange-yellow. Wing under side: ground color yellow-brown, with spots yellow, and blurred.

Female (Fig. 3K). Different from male in reduced spots in spaces R3 and M1, only a pair of small cell spots present on forewing; cell spot and spots in spaces Rs-CuA2 short and small on hindwing.

Male genitalia (Fig. 4A, B). Tegumen slightly shorter than uncus; uncus bifurcated at distal tip with closely aligned arms; gnathos well developed and bifid; valva long and broad, dorsodistal process triangular, ventrodistal process broad and blunt, rounded with small spines at apex, posterior angle ossified but not prominent; lateral process of phallus long, gradually enlarged in distal half, with a row of small spines along dorsal margin; juxta horseshoe shaped.

Female genitalia (Fig. 5A). Papilla analis irregularly triangular in dorsal view; lamella antevaginalis trilobed shaped, lamella postvaginalis U-shaped, with upper margin straight and densely covered with fine hairs and spines; ductus bursae thick and short; bursa copulatrix oblong and membranous.

Etymology

The scientific name is a masculine adjective and derived from the Greek word pseudes (meaning false) and the species name ochraceus, referring to their similarity.

Distribution

China (Zhejiang, Anhui).

Ochlodes cryptochraceus Zhu, Fan & Chiba, sp. nov.

Figs 3L–N, 4C, D, 5B

Ochlodes ochracea: Chiba and Tsukiyama 1996: pl 1 figs 20, 22; Cai et al. 2011: 312; Yuan et al. 2015: 513; Wu and Hsu 2017: 1396.

Type material

Holotype : ♂, SCAU_He2605, 1.VI.2019, Shennongjia, Hubei province, P. R, China. Paratypes: 1♂, SCAU_He2678, 20.VI.2021, Miaowangshan, Baoji, shaanxi province, P. R, China, leg. Liping Zhou; 1♂, SCAU_He2680, 13.VI.2018; 2♂, 6.VII.2018; 1♂, 10.VI.2011, Liukan, Hanzhong, Shaanxi province, leg. Liping Zhou; 1♂, 19.VI. 2018; 1♂, 20.VI. 2018; 1♀, 6.VII.2018, Miaowangshan, Baoji, Shaanxi province, leg. Liping Zhou; 1♂, 10.VII.2011; 1♀, SCAU_He2632, 6.VI.2018; 1♀, 10.VII.2011, Heilongtan, Yulin, Shaanxi province, leg. Liping Zhou; 1♂, 12.VI.2018, Huangguan, Ankang, Shaanxi province, leg. Liping Zhou; 1♂, 24.VI.1993, Wanhuashan, Yanan, Shaanxi province, (HC); 1♂, 5.VII.1993, Qinlin, Shaanxi province, (HC); 1♀, SCAU_He2677, 1.VI.2019, Shennongjia, Hubei province; 1♂, 1.VII.2022, Mianyang, Sichuan province; 1♂, Yunnan province, P. R, China, leg. Xiaoling Fan & Min Wang.

Diagnosis

Spots in spaces R1–R5 on forewing upperside long radial, reaching discocellular vein. Male genitalia: lateral process of phallus only distally enlarged. Female genitalia: upper margin of lamella postvaginalis slightly concave.

Description

Forewing length 15 ± 0.5 mm in males and 14–15 mm in females (Fig. 3L–N). Antenna longer than half length of forewing, and black and white stripes extend to club.

Male (Fig. 3L, M). Forewing upper side: ground color dark brown with orange-red spots. Spots in spaces R1–R5 long radial and connected to discocellular vein; spots in spaces M1–CuA2 form a broad band, of which spot in space M1 very small. Cell orange-red; stigma black-grey and thick. Hindwing upper side: ground color same as forewing, central part including cell and spaces Rs-CuA2 orange-red. Wing under side: ground color red-brown, spots yellow and blurred.

Female (Fig. 3N). Different from male in reduced spots in spaces R1, 2 ; only a pair of small cell spots present on forewing upper side; cell spot and spots in spaces Rs-CuA2 orange-yellow on hindwing.

Male genitalia (Fig. 4C, D). Tegumen slightly shorter than uncus; uncus bifurcated at end tip, with closely aligned arms; gnathos well-developed and bifid; valva long and narrow, dorsodistal process narrow and blunt, ventrodistal process irregular rectangular, and rounded with small spines at apex; lateral process of phallus long and distally enlarged with two rows of small spines; juxta horseshoe shaped.

Female genitalia (Fig. 5B). Papilla analis irregularly triangular in dorsal view; lamella antevaginalis trilobed, lamella postvaginalis U-shaped, with upper margin slightly concave and densely covered with fine hairs; ductus bursae thick and long, and bursa copulatrix oblong and membranous.

Etymology

The scientific name is a combination of the prefix crypt (meaning hidden) combined with the species name ochraceus, which refers to the cryptic species of ochraceus. The name is a masculine adjective.

Distribution

China (Shaanxi, Gansu, Hubei, Sichuan, Yunnan).

Acknowledgments

We would like to express our thanks to Blanca Huertas (NHMUK) for examining the types of Pamphila rikuchina and sending their photographs and Masahiro Ohara (HUM, Hakkaido, Japan) for kindly photographing the type of Augiades amipittiformis. We also thank Huilin Han, Jun Wu (both NEFU, Harbin, Heilongjiang, P. R. China), and Siyao Huang (ZFMK, Bonn, Germany) for kindly examining the specimens of Ochlodes ochraceus and taking valuable photographs of adults and male genitalia. Thanks to Houshuai Wang and Shuqing Ji (SCAU, Guangzhou, P. R. China), Liping Zhou, and Kui Song for collecting specimens; Hiroshi Tsukiyama (Chiba, Japan) for valuable comments; Shinpei Matsuda and Yoshinobu Uemura for the information of O. rikuchina; and Editage for helpful English language editing. We are grateful to the Cave Biology Laboratory of South China Agricultural University for taking photos of genitalia.

Additional information

Conflict of interest

All authors declare that they have no financial or non-financial conflicts of interest.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the National Nature Science Foundation of China (Grants No.31872264 and 31471984 to XLF and 32000326 to ZFH) and by the Ministry of Science and Higher Education of the Russian Federation (Grant No.075-15-2021-1069 to SYS).

Author contributions

Conceptualization: MW, XLF. Formal analysis: LJZ. Funding acquisition: XLF, ZFH. Investigation: LJZ, YXH. Resources: YO, HC, SYS. Supervision: XLF, MW. Original draft writing: LJZ. Review and editing: XLF, MW, HC, YO, YXH, ZFH, SYS.

Author ORCIDs

Lijuan Zhu https://orcid.org/0000-0002-4525-7438

Yongxiang Hou https://orcid.org/0000-0002-4802-9406

Hideyuki Chiba https://orcid.org/0000-0001-9060-6441

Yohei Osada https://orcid.org/0000-0002-0179-9622

Sergey Yu. Sinev https://orcid.org/0000-0002-2467-5403

Min Wang https://orcid.org/0000-0001-5834-4058

Xiaoling Fan https://orcid.org/0000-0002-1176-7667

Data availability

All of the data that support the findings of this study are available in the main text.

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