Systematics of the genus Zinaida Evans, 1937 (Hesperiidae: Hesperiinae: Baorini)

Traditionally, species of the genus Zinaida were assigned to the genus Polytremis, until molecular evidence revealed that the former is a distinct genus. Nine species in Polytremis sensu Evans have since been removed and assigned to Zinaida; however, there is still uncertainty as to the taxonomic status of an additional seven Polytremis species. Moreover, the interspecific relationships within Zinaida have remained unresolved. To further investigate the taxonomic statuses and interspecific relationships within Zinaida, a molecular phylogeny of most species of Zinaida and its allies was inferred based on regions of the mitochondrial COI-COII and 16S and nuclear EF-1α genes (3006 bp). The results revealed that Zinaida is monophyletic and consists of four intra-generic clades that correspond to morphological characteristics. Clade A (Z. suprema group) consists of P. kiraizana, Z. suprema, and P. gigantea, with the latter two as sister species. Clade B (Z. nascens group) consists of seven species, and is the sister group of Clade C (Z. pellucida group), which comprises sister species Z. pellucida and Z. zina. In Clade B, Z. caerulescens and Z. gotama, and Z. theca and Z. fukia are sister species, respectively. On the basis of our molecular evidence and morphological features, we have moved P. gigantea, P. kiraizana, P. jigongi, and P. micropunctata to the genus Zinaida as new combinations. We review morphological characteristics and discuss the distribution of each of these groups in the light of our phylogenetic hypothesis, and provide a comprehensive taxonomic checklist.


Introduction
Recently, the molecular phylogeny of the tribe Baorini, particularly that of the genus Polytremis sensu , has attracted researchers' attention [1][2][3]. Jiang et al. [2] treated Polytremis as a monophyletic genus. In their tree, all the "Polytremis" taxa were nested within a single clade, and P. lubricans, the type of Polytremis, was placed at the distal part of the tree with eltola and, then, those two taxa with discreta. Zhu et al. [3] indicated polyphyly of Polytremis, where lubricans was far apart from the rest of the taxa, but no taxonomic change was made. Fan et al. [1] insisted that the group should be polyphyletic, and divided it into three genera, Polytremis, Zinaida, and Zenonoida. Accordingly, Polytremis was located separately from Zinaida and Borbo is closer than their relationship to Zinaida and Zenonoida. On the other hand, Zenonoida nested with Zenonia and Zinaida, and Zenonia + Zenonoida as sister to Zinaida, whereas Jiang et al. [4] preserved the monophyly of Polytremis. Of the 12 species Fan et al. analyzed, two (P. discreta and P. eltola) were assigned to the newly described Zenonoida, whereas nine were moved to the reinstated genus Zinaida. The only species remaining in Polytremis was its type, P. lubricans. As a consequence, the taxonomic status of seven species of Polytremis sensu  was left uncertain. On the basis of the results of a preliminary morphological analysis conducted as part of the present study, we suggest that P. gigantea Tsukiyama, Chiba & Fujioka, 1997; P. micropunctata ; P. jigongi Zhu, 2012;and P. kiraizana Sonan, 1938 should be included in the genus Zinaida. Our first aim in the present study was to clarify the taxonomic status of these species based on molecular data.
In addition to the aforementioned uncertainty at the generic level, the interspecific relationships within the genus Zinaida also remain unresolved. In previous studies [1][2][3], the sister relationship between Z. zina and Z. pellucida has been the only matter of consensus, whereas the other interspecific relationships have remained uncertain. Accordingly, our second aim in the present study was to investigate the interspecific relationships within Zinaida, and then review the morphological characteristics of these species in light of our phylogenetic hypothesis and provide a comprehensive taxonomic checklist.

Taxon sampling
A total of 17 specimens were examined in this study, seven of which (two Zinaida and five Polytremis, sensu Evans) were newly sampled as ingroup taxa (S1 Table). One of these specimens (He324) appeared to have the male stigma of P. micropunctata and male genitalia of Z. nascens. The specimen was identified as Z. nascens (S1 Fig), which indicates that a difference in the male stigma should not be considered a definitive taxonomic character. Samples of P. micropunctata, P. minuta, P. annama, and P. kittii could not be obtained, and thus their taxonomic status remains unresolved.

DNA extraction, amplification, and sequencing
Samples of Z. matsuii (He1050), P. gigantea, and Z. nascens (He324) were analyzed afresh. The specimens used for DNA analysis were collected as adults and either preserved in 95% ethanol or dried. Total genomic DNA was extracted from dried legs or thoracic tissue using a Hipure Insect DNA Kit (Magen Inc., Guangzhou, China) following the manufacturer's protocol for animal tissues, and stored at -20˚C. Most of the voucher specimens and their extracted genomic DNA were deposited in the Insect Collection, Department of Entomology, South China Agricultural University (SCAU).
For molecular markers, we selected two mitochondrial regions (COI-COII and 16S) and one nuclear locus (EF-1a), in accordance with Fan et al. [1], and added nine new sequences. Moreover, we amplified and sequenced an additional part of the COI region using primers LCO1490 and HCO2198. The remainder of the data was obtained from previous studies [1][2][3] (S1 Table). Methods for the editing and alignment of sequences are detailed in Fan et al. [1] and Huang et al. [5].

Phylogenetic analyses
Species of four Baorini genera-Iton, Zenonia, Zenonoida, and Polytremis-were selected as outgroup taxa, following Fan et al. [1]. The former three genera were closer outgroups, whereas Polytremis, sensu Evans, includes all species of Zinaida in many works.
Phylogenetic trees were constructed using both maximum likelihood (ML) and Bayesian inference (BI) methods. Prior to these analyses, PartitionFinder V1.1.1 was used to select optimal partitioning scheme [6], and the concatenated dataset was treated with the same partitioning scheme for ML and BI analyses (S2 Table). ML analysis was carried out using IQ-TREE [7] on the online W-IQ-TREE (http://iqtree.cibiv.univie.ac.at/, [8]), and the best-fit models for each partitioned dataset (S2 Table) were selected using the Auto function under the Bayesian Information Criterion (BIC) [9]. We performed 1000 ultrafast bootstrap replicates [10] to evaluate branch support (BP). BI analysis was implemented in MrBayes on XSEDE 3.2.6 [11] using reversible-jump MCMC to allow for sampling across the entire substitution rate models. Four Markov Chains (three heated chains, one cold) were run for 2×10 6 generations, sampling at every 1000 generations, and discarding the first 25% of sampled trees as burn-in. Bayesian posterior probabilities (PP) were calculated by majority rule consensus from the remaining trees. Tracer 1.6 [12] was used to examine the programs and determine the convergence of the analyses. MrBayes run was carried out on the online CIPRES Science Gateway resource [13] Morphology Examined samples were collected from China, Japan, Indonesia, and Malaysia (see S1 Table and the checklist for details). For the morphological study, we followed the methods in Fan et al. [14].

Sequence characterization
Twenty-one specimens from five genera were included in this analysis. Twenty-one sequences for COI-II, 19 sequences for EF-1a, and 17 sequences for 16S were characterized. The final combined sequences consisted of 1413 COI-II, 1066 EF-1a, and 527 16S. For the dataset, 559 bp sites were variable, and 323 sites were parsimony-informative.

Phylogenetic analysis
The same tree topology was obtained using both ML and Bayesian methods (Fig 1). Polytremis, as circumscribed by many authors [3,[15][16][17], is not monophyletic, as shown by Fan et al. [1]. The monophyly of the genus Zinaida is strongly supported (BP = 96, PP = 1.00). It is divided into four clades, namely the Z. matsuii clade, Clade A (BP = 98, PP = 1.00), Clade B (BP = 99, PP = 1.00), and Clade C (BP = 100, PP = 1.00). Clade B is sister to Clade C, whereas it is not entirely clear whether the clade comprising Z. matsuii and Clade A are sisters to each other, or whether one or the other is sister to Clades B + C, due to the lack of strong statistical support (BP = 72, PP = 0.69). Regardless, P. gigantea, P. jigongi, and P. kiraizana were shown to belong to the genus Zinaida.
Clade A consists of Z. kiraizana, Z. suprema, and Z. gigantea, and relationships between the three species were well resolved as [Z. kiraizana + (Z. suprema + Z. gigantea)]. Clade C consists of Z. pellucida and Z. zina, and is strongly supported in our analyses. The remaining eight species were included in Clade B, and relationships between some of these species were well resolved; for example, Z. gotama is sister to Z. caerulescens, and Z. jigongi is sister to Z. nascens.

Phylogeny of the genus Zinaida
The phylogenetic framework including nearly all the species of the genus Zinaida and its allies revealed four distinct clades that are consistent with their external features, although the relationships between the four groups were not entirely clarified. In this study, the monophyly of the Z. suprema group (Clade A: Z. suprema, Z. gigantea, and Z. kiraizana) is strongly supported. These species share the following synapomorphies: (1) aedeagus with suprazonal sheath bifurcated into serrated symmetrical processes (processes asymmetric in Z. matsuii), and (2) cornuti absent (Fig 2A-2D). Z. suprema is a sister species to Z. gigantea, and the relationship is supported by overall similarity in male genital characteristics (Fig 2C and 2D). This species group, as well as Z. matsuii, share the following similar morphological characteristics: (1) forewing with two cell spots, lower one wedge-shaped, pointing toward wing base (Fig 1: 1-4); (2) uncus, left and right projections U-shaped; (3) saccus long and thin; (4) aedeagus, more than basal 2/3 of subzonal sheath thin (Fig 2A-2D). In the analyses of Jiang et al. [2], Z. suprema, Z. gigantea, Z. kiraizana, Z. matsuii, and Z. caerulescens formed a well-supported clade. However, it is obvious that Z. caerulescens does not belong to this species group. Fan et al. [1] and Zhu et al. [3] reclaimed the close association of Z. matsuii and Z. suprema, and that of Z. matsuii and Z. kiraizana. Interestingly, the three species have a similarly shaped male stigma in space CuA2 on the upper side of the forewing (Fig 1: 1-3). On the basis of our present phylogenetic inference and previous studies, as well as morphological characteristics, we recognize that the Z. suprema group consists of Z. kiraizana, Z. suprema, and Z. gigantea. For Z. matsuii, because of the low statistical support and a morphological difference (asymmetric suprazonal sheath of the aedeagus), further study is necessary to confirm its association with this clade.
Fan et al. [1] showed a well-supported clade that comprised Z. caerulescens, Z. nascens, Z. gotama, Z. mencia, Z. theca theca, and Z. theca fukia, although interspecies relationships were not clarified. Our phylogenetic inferences confirm that these species form a monophyletic group (Clade B), the Z. nascens group, within which P. jigongi is also nested. This analysis also revealed that P. caerulescens and Z. gotama are sister species, and placed P. jigongi as a sister to Z. nascens. Accepting the conclusion of Jiang et al. [4] that Z. theca and Z. fukia are distinct species, the Z. nascens group proposed in this analysis includes the above seven species, which share some notable morphological characteristics: (1) lower cell spot on the forewing not produced toward base, or no spot at all (Fig 1: 5-11); (2) aedeagus with suprazonal sheath bifurcated distally; and (3) uncus V-shaped, left and right projections closer (Fig 2E-2J), and show the following relationships: (mencia + ((theca + fukia) + ((jigongi + nascens) + (gotama+caerulescens)))). Morphologically, Z. mencia, Z. theca, Z. fukia, and Z. jigongi have two cell spots on the forewing, but the remaining three, Z. nascens, Z. gotama, and Z. caerulescens only have an upper cell spot or no spot. Z. gotama and Z. caerulescens have no cornuti, whereas in the remaining five species cornuti are present. Moreover, in Z. theca and Z. fukia, the ventrodistal process of the valva is distally short, blunt, and unbifurcated. On the basis of the morphological characteristics detailed in the original description [18], P. micropunctata belongs to this group and is very probably a sister species of Z. nascens.
The Z. pellucida group (Clade C) consists of Z. pellucida and Z. zina. A close association between the two species was also identified in previous studies [1][2][3]. Our morphological study shows that they share the following morphological characteristics: (1) large spots in space M2-CuA1, with the distance between them shorter than in any other species (Fig 1: 12-13); (2) uncus short, left and right projections separated, gnathos far longer than uncus; and (3) aedeagus with suprazonal sheath slightly bifurcated distally or not bifurcated, cornuti absent ( Fig  2K, l).
Our analysis lacks these specimens of Polyremis minuta, P. annama, and P. kittii. On the basis of the morphological characteristics detailed in the original description [15,19,20], these species share the following synapomorphies: (1) the third segment of palpi short, stout and barely protruding; (2) forewing cell spots conjoined or only under cell spot present. Therefore, we tentatively assign them to Zenonoida. Further study is necessary to confirm this hypothesis, and clarify their phylogenetic placement.

Distribution
Most species in the genus Zinaida are endemic to China. Each of the identified clades shows a large degree of similarity in terms of wing pattern and male genitalia characteristics, and exhibits an interesting geographical pattern. The Z. suprema group and Z. matsuii have narrower ranges: Z. kiraizana is only found in Taiwan, whereas the other three species: Z. suprema, Z. gigantea, and Z. matsuii currently occur in Zhejiang, Fujian, Guangdong, Guangxi, Sichuan, Guizhou, and Yunnan [16,17,21] (Fig 3A). The sister species Z. suprema and Z. gigantea are sympatrically distributed, and have been found simultaneously in Nanling, Guangdong. Conversely, Z. kiraizana and the former two species (Z. suprema and Z. gigantea) have allopatric distributions, suggesting geographical isolation caused by the strait between the Chinese mainland and Taiwan that occurred earlier than the speciation of Z. suprema and Z. gigantea.
All species in the Z. nascens group are endemic to China, and mainly occur south of the Qinling Mountains and east of the Hengduan Mountains (Fig 3B). According to the known distribution data, the center of diversity of the group is central Sichuan (Yaan and Kangding), which is inhabited by five Zinaida species: Z. caerulescens, Z. nascens, Z. theca, Z. fukia, and Z. mencia. These species do extend their range to other regions. Z. mencia and Z. fukia have the broadest geographical ranges, including southwest China and subtropical (central/east/south) China. In our present analyses, they represent the basal branches of the group. The other species are mainly distributed in southwest China and adjacent regions. Notably, the sister species Z. theca and Z. fukia, having been treated as subspecies of P. theca  previously [15], have a narrow geographical overlap in central Sichuan, China [4]. Z. caerulescens is distributed in Chongqing, Sichuan, Guizhou, Yunnan, and Xizang, and shows an overlap with Z. gotama in North Yunnan. Interestingly, these two sympatric species are sister species according to the molecular analysis. Clearly, the species are not evenly distributed, and closely follow the distributions of certain topographic features and climatic conditions. The diversity of the group, as well as that of other Chinese skippers, is greatest in Southwest China [22], which is considered one of the world's biodiversity hotspots because of its meteorological heterogeneity, monsoons, and specific topography, comprising a mosaic of plateaus, mountains, basins, river valleys, and deep gorges [23][24][25].
Finally, Z. pellucida and Z. zina have a broader geographic range than other Zinaida species, and have extensive range overlap in Southeastern China, as well as in North Korea (Fig 3C).  uncus, left and right projects attached at base, attached to straight gnathos; suprazonal sheath of aedeagus usually bifurcated into serrated processes.

Taxonomic checklist
The genus externally very similar to Polytremis and Zenonoida, can be distinguished from former by wing white spots, undivided lateral process of uncus; and separated from latter by thin, long, prominently protruding third segment, forewing cell spots separated or lower cell spot absent.
Supporting information S1 Table. List of species used in this study. (DOCX) S2 Table. The