﻿Molecular and morphological evidence reveals hidden new taxa in Ochlodesochraceus (Bremer, 1861) (Lepidoptera, Hesperiidae, Hesperiinae) from China

﻿Abstract Two new species of Ochlodes Scudder, 1872, Ochlodespseudochraceus Zhu, Fan & Wang, sp. nov. and Ochlodescryptochraceus Zhu, Fan & Chiba, sp. nov., are found in China and described, and Ochlodesrikuchina (Butler, 1878) stat. rev. is restored. A lectotype is designated for Pamphilaochracea Bremer, 1861, and a neotype is designated for Pamphilarikuchina Butler, 1878. Overall, the two new species are similar to Ochlodesochraceus (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.


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 (1949Evans ( , 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 classi-

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 × 10 6 generations simultaneously, sampling every 1000 generations, with the first 25% of sampled trees discarded as burn-in. 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.

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 R 3 -R 5 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 CuA 1 and the second in space CuA 2 are markedly different, whereas the third part in space CuA 2 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 spe-  (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 4 th and 5 th 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.

Taxonomic accounts
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 R 1 -R 5 long radial and connected to discocellular vein; spots in space M 1 to CuA 2 form a broad brand, of which spot in space M 1 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-CuA 2 orange. Wing under side: ground color orange-yellow, with spots orange, and blurred.
Female (Fig. 3G). Different from male in reduced spots in spaces R 1 , 2 ; only a couple of cell spots present on forewing; cell spot and spots in spaces Rs-CuA 2 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.
Distribution. Japan (Honshu, Shikoku and Kyushu). Diagnosis. Spots in spaces R 3 -R 5 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.

Ochlodes pseudochraceus
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 R 3 -R 5 short and radial, far from discocellular vein; spots in space M 1 to CuA 2 form a broad brand, of which spot in space M 1 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-CuA 2 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 R 3 and M 1 , only a pair of small cell spots present on forewing; cell spot and spots in spaces Rs-CuA 2 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.
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.

Ochlodes cryptochraceus
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 R 1 -R 5 long radial and connected to discocellular vein; spots in spaces M 1 -CuA 2 form a broad band, of which spot in space M 1 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-CuA 2 orange-red. Wing under side: ground color red-brown, spots yellow and blurred.
Female (Fig. 3N). Different from male in reduced spots in spaces R 1 , 2 ; only a pair of small cell spots present on forewing upper side; cell spot and spots in spaces Rs-CuA 2 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.
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.