Discovery of a peculiar insular race of Ravenna nivea (Nire, 1920) (Lepidoptera: Lycaenidae) endemic to Yinggeling Mountain of Hainan, suggesting heterogeneous geological history of mountain formation of the island

Acquisition of material

Adult butterflies in question were collected from mountain slopes of Yinggeling, Hainan (109°11′27″–109°34′06″, 18°49′30″–19°08′41″), China. Ova were found near dormant buds of the hostplants. Larvae were provided with foliage of the hostplants. Rearing was performed using plastic containers 8 × 5.5 × 3 cm in size, with each larva kept separately.

Besides samples of Ravenna collected from Yinggeling, specimens of R. nivea from the following localities were examined and compared for morphological characters: ssp. nivea, 7♂13♀ (Taiwan); ssp. howarthi, 16♂22♀ (2♂2♀, Guangdong, 4♀, Jiangxi, 1♂2♀, Hainan [Bawangling], 1♂1♀, Fujian, 2♂, Zhejiang, 3♂5♀, Sichuan, 7♂8♀, Guizhou); ssp. miyagawai, 4♂5♀ (Vietnam). Of them, exemplars of taxa used for sequencing of the COI barcode are given in Table S1. COI barcodes of Leucantigius atayalicus and Yamamotozephyrus kwangtungensis were obtained from NCBI as reference outgroups (Table S1). Ssp. koiwayai, characterized by a reduction of tornal orange markings on hindwing undersides (Yoshino, 1997), is only known from its type locality, Mt. Konga of Sichuan in western China (Koiwaya, 2007), and was not available for the present study. The voucher specimens used for comparison listed above are currently stored in Department of Life Science, National Taiwan Normal University, Taipei (NTNU). The distribution of the samples used in the present study is shown in a map created using the Free and Open Source QGIS. Hainan Wildlife Conservation Bureau, Yinggeling National Nature Reserve and Bawangling National Nature Reserve granted permission to conduct the survey and provided essential assistance with field work.

Molecular work sampling

Butterfly identification was conducted based on morphological characteristics. The tissue samples were preserved in 95% ethanol and stored frozen at −80 °C for further DNA extraction. Taxa listed in Table S1.

DNA extraction and sequencing

Tissue from two adult legs was digested using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The COI gene was amplified by PCR using a set of primers (Table S2). The amplification program was: 5 min at 94 °C, 40 cycles of 30 s at 94 °C, 30 s at 45–50 °C, and 1 min at 72 °C, and a final elongation step of 10 min at 72 °C. The PCR products were run on 1.0% agarose gels in 1x TBE buffer to ensure correct amplification. PCR products were cleaned using a Gel/PCR DNA Fragments Extraction kit (Geneaid, Taipei, Taiwan) when only a single DNA band was visible in a gel. Sequencing reactions were conducted using a 96-well Gel/PCR Clean Up kit (Geneaid) on an ABI3730XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA). All DNA sequences have been submitted to the NCBI GenBank and accession numbers are given in Table S1.

Molecular data analyses

Molecular sequences of COI gene were checked and assembled into contigs using Sequencher 4.10 (GeneCode, Boston, MA, USA). The software MEGA6 (Tamura, Filipski & Kumar, 2013) was used to perform sequence alignment using the MUSCLE method and sequence divergence using the Kimura2-Parameters (K2P) method. The partition schemes were determined with PartitionFinder v.2.1.1 (Lanfear & Calcott, 2017). The data matrices were analysed using maximum likelihood (ML) with RAxML and Bayesian inference (BI) with MrBayes on CIPRES (Miller, Pfeiffer & Schwartz, 2010). Leucantigius atayalicus and Yamamotozephyrus kwangtungensis were chosen as the outgroup because the former is considered sister to Ravenna by Hsu & Chou (2001) and the latter to be closely related to Ravenna by Koiwaya (2007). The program MrBayes 3.2.6 (Ronquist et al., 2012) was used simultaneously for 5 million generations. We removed the first 25% burn-in parts and used the remainder to generate a 50% majority consensus tree. Assessing the effective sample size (ESS), evaluating the parameters, and estimating convergence of two runs were performed with the software Tracer v.1.7 (Rambaut et al., 2018). Phylogenetic trees were read by FigTree v.1.4.3 (see http://tree.bio.ed.ac.uk/software/figtree/). The software program DnaSP 5.10.01 (Rozas et al., 2017) was used to calculate genetic parameters including the nucleotide composition, the number of polymorphic sites, and variable nucleotide positions. A haplotype network was generated via median-joining method using POPART 1.7 (Leigh & Bryant, 2015). Haplotype networks were constructed using the TCS analysis in the POPART software.

Material depository

Primary types are deposited in the following collections: Institute of Zoology, Academia Sinica, Beijing (IOZ), Kadoorie Farm and Botanic Garden, Hong Kong (KFBG), and Department of Biology, National Taiwan Normal University, Taipei (NTNU).

Terminology for description

Measurements are defined and abbreviated as follows: forewing length (FL) and antennal length (AL). Terminology of wing patterns follows that of Nijhout (1991).

Nomenclature

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:434949A4-F758-4C1D-A332-B2D2E8906098. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE and CLOCKSS.

Genetic divergence

The genetic divergence of COI barcode found in the samples used in the present study is shown in Tables S3 and S4. The phylogenetic tree based on BI analysis is given in Fig. 2. The ML tree was slightly different from the BI tree in topology, but all groupings with nodes of bootstrap values over 75% were conformed to the later (Fig. 2). DNA barcode sequences (1,034 bp) were obtained from 80 samples (Table S1). A total of 37 variable sites were detected, with 24 haplotypes defined. Two out of 37 polymorphic sites were singleton variable sites, one from Yinggeling and one from Zhejiang. The variation of mtDNA revealed the presence of 24 haplotypes, seven found only in Taiwan, three restricted to Yinggeling, seven to Bawangling, four to Zhejiang, and two to Vietnam. Samples from Guizhou and Guangdong shared the same haplotype, and those from Jiangxi and Fujian had the same haplotype. The result of the TCS analysis is shown in Fig. 3. The overall haplotype diversity (Hd) was 0.899 and nucleotide diversity (π) was 0.012. The values of haplotype diversity and nucleotide diversity that were present are shown in Table S3. The haplotype diversity of Ravenna nivea from Yinggeling and Bawangling within Hainan are high, suggesting that the genetic variance is associated with geographic distribution and isolation.

Taxonomic decision

The pairwise genetic divergence of the COI barcode was 0–1.7% between Ravenna samples examined from different sites and 1.1–1.5% between the Yinggeling sample and samples from the other localities (Table S4), both of which are lower than the 3% COI genetic divergence for species discrimination of Lepidoptera suggested by Hebert et al. (2003). According to Braby, Eastwood & Murray’s (2012) scheme for testing hypothesis of taxonomic status of allopatric populations, the null hypothesis of a single species is rejected only if evidence from other multiple data sources (color pattern, morphology, behavior, ecology, genetics, etc.) supports the alternative. The morphology of immatures of the Yinggeling (Fig. 4) is indistinguishable from that of R. nivea from the other localities (see p. 481 in Igarashi & Fukuda, 1997; p. 33 in Uchida, 1999; fig. 45 in Koiwaya, 2007; P. 223 in Lu & Chen, 2014) in morphology and biology. There is also no conceivable diagnosis present in morphology of genitalia of the Yinggeling samples (Figs. 5, 8A) from those of the other subspecies (Figs. 6, 7, 8B, 8C). The morphology, biology, and COI barcodes thus render support to place the Yinggeling Ravenna sample within R. nivea, but deserving a subspecies status with its well-differentiated wing patterns and genetic make-up. Accordingly, a new subspecies is described herein.

Systematics

Type materials. Holotype. ♀: CHINA: Hainan Prov., Baisha Xian, Yinggeling, 1,200–1,300 m, 30. I. 2015, reared from Qercus fleuryi, emgd. 4. III. 2015, Coll. Y. F. Hsu & Y. F. Lo (HSU 15A29) (IOZ).

Paratypes. 5♀: same locality as for holotype, 1,000–1,400 m, 13. V. 2014, Coll. Y. F. Lo (4♀, KFBG; 1♀, NTNU); 1♂3♀, same collecting data as for holotype, emgd. 26. II. –7. III. 2015 (HSU 15A29) (1♂ dissected, genitalia preparation YFH 1565, IOZ; 1♀, NTNU; 2♀, KFBG) (DNA voucher YFH 170160); 1♂, same locality as for holotype, 1,200–1,300 m, 30. I. 2015, reared from Q. hui, emgd. 9. III. 2015, Coll. Y. F. Hsu & Y. F. Lo (HSU 15A34) (NTNU); 2♂3♀, same locality as for holotype, 1,200 m, 31. I. 2016, reared from Q. fleuryi, emgd. 7–27. III. 2016 (HSU 15A40) (1♂ dissected, genitalia preparation YFH 1561, NTNU; 1♀, NTNU; 1♂2♀, KFBG); 1♂1♀, same locality as for holotype, 1,500 m, 31. I. 2016, reared from Q. hui, emgd. 24. III/19. IV. 2016 (HSU 15A50) (NTNU).

Description. Male (Figs. 1E–1F): FL 19.2–22.0 mm (mean, 20.4 ± 1.1 mm, n = 5); AL 8.3–8.7 mm (mean, 8.6 ± 0.2 mm, n = 4). Head: Hairy, vertex, frons dark brown, with mesal white patch on vertex.

Eye semi-oval, sparsely covered with short, buff setae, surrounded by a narrow white rim. Labial palpus porrect, with 3rd segment pointed downwards, covered with white scalings and hairs, dark brown apically. Maxillary palpus reduced, inconspicuous. Proboscis unscaled. Antenna smoothly scaled, naked at terminal end of nudum, dark brown with lateral white dots on each flagellomere. Thorax: Dark brown dorsad, white ventrad. Legs with tarsus of foreleg segmented; white, with brown bands. Forewing: Generally triangular in shape, with apex slightly obtuse; termen, costa slightly convex; dorsum straight. Ground color of upperside purple, with narrow, dark brown margin along termen. Fringe with outer cilia white, inner cilia brown. Ground color of underside white. Discal spot as hollow brown bar. Distal band of central symmetry system represented as a pair of well-separated brown line, approximate in cell Cu₂. Submarginal band as a series of faint, brown spots; element g (sensu Nijhout, 1991) as a broken brown band. Fringe white, prominent. Hindwing: Contour of wing slightly produced at distal end of Cu₁; Cu₂ bearing long, tail-like projection. Ground color purple, with brown area along costa; faint, narrow, white margin along termen. Fringe white. Ground color of underside white. Discal spot inconspicuous. Distal band of central symmetry system represented as a pair of brown line, approximate posteriad, dislocated proximally as tick-shaped lines in cell Cu₂, retracing into straight lines in cell 1A+2A. Proximal band of central symmentry as a broken, brown line. Submarginal band and element g similar to those of forewing, but a black, rounded spot crowned with orange in cell Cu₁ and at tornus. Fringe white, prominent. Abdomen: Brown dorsally, white ventrally. Genitalia (Fig. 5): Ring-shaped sclerites of 9+10 segments with posterior end quadrifurcate. Tegumen broad. Socii prominent, short, conical, setose. Uncus forming a pair of elongate, lateral processes with pointed distal tip, slightly down-curved. Vinculum narrow. Brachia (falces) up-curved, ox horn-like, distal end pointed. Saccus longer than length of tegumen, with anterior end obtuse. Valva elongate, narrowed posteriorly with distal end rounded, small teeth present along inner margin. Juxa broad ventrally, with two arms extending dorsad, forming opposite pointed tips. Phallus with phallobase approximately as long as aedeagus. Aedeagus with dorsal opening at distal end, blunt at caudal tip.

Female (Figs. 1G, 1H). FL 19.0–21.7 mm (mean, 20.4 ± 1.1 mm, n = 13); AL 7.0–8.8 mm (mean, 8.1 ± 0.6 mm, n = 13). Body, wing patterns of underside as described for male except ground color darker. Wing upperside mostly white with extensive purple scalings proximally. Prominent dark brown scalings covering forewing apex, along costa and termen, and along termen of hindwing. Prominent dark brown bar present at distal end of discoidal cell of forewing; faint, narrow, white margin also present along termen of hindwing. Genitalia (Fig. 8A): Corpus bursae oval, elongate. Ductus bursae short, thick, sclerotized and enlarged posteriorly, with large ostium bursae. Sterigma with lamella antevaginalis forming a pair of sclerotized, lateral patches; lamella postvaginalis as broad sclerotized wall, with posterior margin produced, blunt. A small, detached transverse, rectangular, sclerotized band posterior to lamella postvaginalis. Posterior apophyses slender, much longer than anal papillae, enlarged and flattened basally. Anal papillae as weakly sclerotized band, setose.

Immatures. Egg (Fig. 4A) 0.82 ± 0.03 mm in diameter, 0.49 ± 0.02 mm in height (n = 15), hemispherical but compressed, white, surface with sculpture forming framework with regularly arranged, stub-like processes. Larvae (Figs. 4C, 4D) with four instars. Head brown, glossy. Body onisciform, with surface bearing transparent setae; T1 shield as a short, transverse band, slightly produced laterally; anal lobe semi-circular, with posterior margin rounded. Ground color of body green tinged with yellow. Red markings present in late instars, laterally and around medial and caudal portion dorsally. White chevrons present subdorsally. Spiracles white, surrounded by brown peritremes. Full-grown larva (Fig. 4D) reaching 18 mm in body length. Pupae reaching 12 mm in length, of typical lycaenid form, ground color pale brown, decorated with yellow chevrons and dark brown, subdorsal bands; spiracles cream white.

Diagnosis. The taxon ngiunmoiae ssp. nov. can be distinguished from other subspecies of R. nivea by the following external features: (1) in the males, prominent white scalings are absent on wing uppersides of ngiunmoiae (Fig. 5), whereas present -in the other subspecies (Fig. 1), but; (2) in the females, extensive purple scalings are present on wing upperside in ngiunmoiae (Fig. 7), whereas weakly-developed brown scalings are found proximally in the other subspecies (Fig. 3).

Hostplants. Quercus hui and Q. fleuryi (Fagaceae).

Distribution. Known from montane zone of Mt. Yinggeling, central Hainan approximately 1,000–1,500 m in elevation.

Phenology. Univoltine. Adults were collected in May, suggesting occurrence of adult imago may be in early summer. Overwintering as egg near bases of dormant buds on the hostplants.

Etymology. The subspecific name ngiunmoiae refers to the pronunciation of the name of the late grandmother , in the Hakka dialect, of the first author of the new subspecies, YFP Lo, for her warm and full support to his scientific work on butterflies and other animals.