A review of tiger moths (Lepidoptera: Erebidae: Arctiinae: Arctiini) from Flores Island, Lesser Sunda Archipelago, with description of a new species and new subspecies

The Wallacean Region is considered a unique evolutionary hotspot, but the current knowledge of lepidopteran faunas on certain islands is very far from being complete. Here we present a preliminary checklist of the Arctiini fauna of the Flores Island based on available collection materials and a review of the body of literature. In total, for the island fauna we list 22 tiger moth species, with eight newly recorded species. Among novel records, local endemic Spilarctia mikeli Bolotov, Kondakov & Spitsyn sp. nov. and Aloa cardinalis danau Bolotov, Kondakov & Spitsyn ssp. nov. were discovered. Additionally, several taxa with broad ranges such as Amerila astreus, Creatonotos gangis, Euchromia horsfieldi, Lemyra maculifascia, Nyctemera distincta and Utetheisa pulchelloides were new for the island fauna. In general, 15 taxa are prospective endemic Wallacean elements, among which seven taxa are unknown outside the Flores Island: Orhantarctia cymbalophoroides, Lemyra everetti, L. floresina, Aethalida owadai floresiensis, Nyctemera scalarium regalis, Spilarctia mikeli sp. nov. and Aloa cardinalis danau ssp. nov. Our findings reveal that the tiger moth fauna of the Flores Island has rather moderate level of endemism, with only 32% of putative endemic taxa.

The present investigation aims to review the current knowledge of the Arctiini fauna of the Flores Island in a broad biogeographic context.Based on this work, we perform the first but still incomplete checklist of this fauna and describe two taxa new to science, i.e., a new remarkable species in the genus Spilarctia Butler, 1875 and a new local subspecies of Aloa cardinalis (Butler, 1875).

Material and Methods
This study is based on available materials from the Lepidoptera collection of the Russian Museum of Biodiversity Hotspots, Federal Center for Integrated Arctic Research, Russian Academy of Sciences, Arkhangelsk, Russia (RMBH thereafter), the Natural History Museum, London, United Kingdom (BMNH) and Naturalis Biodiversity Center, Leiden, The Netherlands (RMNH) (Tables 1 and 2).Additionally, we reviewed published occurrences of tiger moths from the Flores Island (Table 1).
The genitalia were dissected and mounted on a glass slide with Histofluid® (Paul Marienfeld GmbH & Co., Germany).The images of specimens were taken with a Canon EOS 650D camera (Canon, Tokyo, Japan).The photos of the genitalia were obtained using a research stereomicroscope (SteREO Discovery.V8, Carl Zeiss, Germany).Specimens of Amerila astreus, Creatonotos gangis, and Spilarctia mikeli sp.nov.were studied by using a molecular approach (Table 2).The total DNA was extracted from a single leg of each dry specimen using a standard phenol/chloroform procedure (Sambrook et al., 1989).The standard primers LepF and LepR were used for the amplification of 660-bp-long barcode fragments of the cytochrome c oxidase subunit I (COI) gene (Hajibabaei et al., 2006).The PCR mix contained approximately 200 ng of total cellular DNA, 10 pmol of each primer, 200 μmol of each dNTP, 2.5 μl of PCR buffer (with 10×2 mmol MgCl 2 ), 0.8 units Taq DNA polymerase (SibEnzyme Ltd., Novosibirsk, Russia), and H 2 O added for a final volume of 25 μl.Thermocycling included one cycle at 95°C (4 min), followed by 38-40 cycles of 95°C (50 sec), 50°C (50 sec), and 72°C (50 sec) and a final extension at 72°C (5 min).Forward and reverse sequencing was performed on an automatic sequencer (ABI PRISM® 3730, Applied Biosystems) using an ABI PRISM® BigDye™ Terminator v. 3.1 reagent kit.Resulting sequences were checked manually using a sequence alignment editor (BioEdit version 7.2.5;Hall, 1999).
obtained from these sources.For phylogenetic analyses of Spilarctia spp., we collected a sequence data set with 26 unique COI haplotypes (658 bp in length).As an out-group, a haplotype of Theretra natashae Cadiou, 1995 was used (Table 2).The lacking sites were treated as missing data.The best models of sequence evolution as suggested the corrected Akaike Information Criterion of MEGA6 (Tamura et al. 2013) were as follows: 1 st codon of COI: GTR+G (G = 0.85), 2 nd codon of COI: TN93+G+I (G = 0.41; I = 0.40), and 3 rd codon of COI: HKY.Phylogenetic relationships were reconstructed based on Bayesian inference implemented in MrBayes v. 3.2.6 (Ronquist et al., 2012).The analyses were performed using the following parameters: nchains = 4, nruns = 2, samplefreq = 1000, temp = 0.1; 10% of the sampled trees were discarded as burn-in (pre-convergence part).Runs were conducted for 3 million generations.Convergence of the MCMC chains to the stationary distribution was checked visually based on the plotted posterior estimates using a MCMC trace analysis tool (Tracer v1.6;Rambaut et al., 2014).Calculations were performed at the San Diego Supercomputer Center through the CIPRES Science Gateway (Miller et al., 2010).Distribution: Sumatra, Java, Borneo, Bali, Lesser Sundas, and Christmas Island (Table 1).

Systematics
Remarks: First record from Flores.The lengthened dark yellow spot below the cell on the forewing of our female specimen is separated by large black triangular inclusion (vs.continuous dark yellow spot with only a minute black inclusion in specimens from Java and Borneo: Moore, 1859;Holloway, 1988).
Distribution: India and Sri-Lanka over Nepal, continental China, Taiwan, the Philippines to Indo-China, Indonesia and New Guinea (Table 1).
DNA barcoding: Two sequenced specimens from Flores share two unique COI haplotypes with pdistance of 0.3%.Their nearest neighbors were from Malaysia (p-distance from 0.2 to 0.6%) and from Taiwan (p-distance from 1.1 to 1.4%) (Table 2) that indicates rather recent dispersal of the species from Sundaland to the Lesser Sundas.
Remarks: First record from Flores and its small offshore island (Kanawa).Etymology: Danau = lake in Bahasa Indonesia as the new subspecies was discovered on the shore of the Sano Ngoang Lake.
Diagnosis: The females of West Flores's subspecies differs from A. c. cardinalis (Butler, 1875) from the Philippines and A. c. celebensis (Rothschild, 1910) from Sulawesi by the lack of submarginal spots or presence of 1-2 small, elongated spots on the hindwing (vs.large black submarginal patches); from A. c. reducta (Rothschild, 1910) from the Tomea Island (Tukangbesi Archipelago) by the lack of submarginal spots or presence of 1-2 small, elongated spots on the hindwing (vs. 3 black submarginal spots); and from A. cardinalis luteomarginata (Rothschild, 1910) from Timor, East Flores and Maluku by red coloration on costa, collar and edges of tegulae (vs.yellow).The males of West Flores's subspecies differ by darker creamy or light brown ground color of the hindwing (vs.white).
Description.Male morphology: Wingspan 59-63 mm, forewing length 28-30 mm (N = 3).Labial palpi stout, upright, short (equal to eye diameter).Eyes naked.Antennae long, filiform, thinner apically.Abdomen long.Male markings: Head white, with red dorsal margin.Antennae black.Labial palpi dorsally black, ventrally white or yellow.Eyes gray.Thorax dorsally white.Collar white, with red margin.Tegulae white, with black dot near the base and red edge.Legs dorsally black, ventrally white or yellow.Abdomen dorsally bright red, with a row of black dots; ventral side of abdomen white; a row of large black spots is located dorsally, with two rows of smaller black spots below.Forewing white, costa red, two small black spots near dorsal margin and two small black spots in distal part of cell.Hindwing creamy up to light brown, fringe white, up to 3 small black spots near termen, large black spot in cell.Underside of both wings white, usually with similar markings with black spots as on the upperside.Male genitalia: Asymmetric, differs from the nominative subspecies by a triangular recess on the apical part of the right valva and by two tubercle-like processes on the inner side of the left valva (Fig. 5C-5D).Female morphology: Wingspan 65-73 mm, forewing length 30-35 mm (N = 3).Labial palpi stout, upright, short (equal to eye diameter).Eyes naked.Antennae long, filiform, thinner apically.Abdomen long.Female markings: Eyes gray.Head white, with red dorsal margin.Antennae black.Labial palpi dorsally black, ventrally white, yellow or pinkish.Eyes gray.Thorax dorsally white.Collar white, with red margin.Tegulae white, with black dot near the base and red edge.Legs dorsally black, ventrally white or yellow.Abdomen dorsally bright red, with a row of black dots, which may lacking, and sometimes with a few black spots on the distal part of abdomen; ventral side of abdomen white; a row of large black spots is located dorsally, which sometimes merge into a line, with two rows of smaller black spots below; end of abdomen white.Forewing white, costa red, two small black dots near dorsal margin and two small black dots in distal part of cell.Hindwing creamy, black spots near termen usually lacking but up to 2 spots sometimes present, black spot in cell.Underside of both wings white, usually marking with black spots is reduced, with exception of black spots in cell.Female genitalia: Not examined.
Distribution: West Flores; only known from two localities but most likely distributed across neighboring islands, e.g., Sumbawa and Sumba.

Remarks:
The range of each subspecies of A. cardinalis is confined to a certain archipelago or island that corresponds to the allopatric speciation model (Rothschild, 1910;Dubatolov, 2004;Černý 2011).With respect to this biogeographic pattern, these isolated subspecies may actually represent several divergent species-level lineages but this hypothesis need to be checked in a future on the basis of molecular sequence data.
Distribution: Widespread from Middle East (Oman, the United Arab Emirates and Iran), India and China to Australia (Table 1).
DNA barcoding: Two sequenced specimens from Flores share two COI haplotypes (GenBank acc.nos.KY683800 and KY683801) with the mean p-distance 0.2%.Their nearest neighbors were originated from South Asia (India, Pakistan and Nepal) and the Middle East (Oman) with the minimum p-distance of 1.6% (Table 2).
Remarks: First record from Flores.
Lemyra maculifascia (Walker, 1855) Fig. 2H  Diagnosis.The new species is similar to Spilarctia wahri (Rothschild, 1933) from Timor, S. mindanao Dubatolov & Kishida, 2010[=S. trikenzana (Cerný, 2011)] and S. mollis (Cerný, 2011) from the Philippines, but differs by reduced black markings on the hindwing, with stigma spot, postdiscal dot near the stigma, a row of a few minute postdiscal black dots, and two minute submarginal marks.The female of S. wahri has two black spots near the tornus and two black dots near the apex; the forewings are dark brown (Rothschild, 1933).The female of S. mindanao has lunular black spot in cell and three black spots in submarginal area.The female of S. mollis has black spot in cell and black submarginal band consisting of four partly conjoined patches.
Description.Female morphology: Wingspan 41 mm, forewing length 21 mm.Head with frons equal to eye diameter.Labial palpi stout, upright, short (slightly longer than eye diameter), underside ciliate (with scarce short ciliae).Proboscis small, weakly developed.Antennae long, filiform, thinner apically, with two short ciliae on each segment.Abdomen long.Female markings: Head and antennae brown.Labial palpi brown with black end.Thorax dorsally brown.Underside of thorax red-brown.Legs light orange-pink.Tegulae brown.Abdomen dorsally bright rose-red with large, rounded black spots on each tergite, distal end brownish black.The ventral side of the abdomen yellow pink.Forewing light brown, with rows of small, unclear blackish spots in postbasal, postdical and postmarginal areas, and two black spots at the base of the wing.Hindwing bright rose-red, black markings strongly reduced, with rounded stigma spot, one postdiscal spot near the stigma, a row of a few minute postdiscal black dots athwart to the anal margin, and two submarginal marks.Underside of both wings orange-pink, with rows of black postdiscal and submarginal spots.Black discal spot on each wing.Female genitalia: Not examined.
Male is unknown.DNA barcoding: Reference COI sequence no.MG735265.There are no available nearest members in GenBank and BOLD IDS.The new species looks morphologically similar to taxa in the subgenus Praephragmatobia Dubatolov & Kishida, 2010, but it is genetically distant from Spilarctia strigatula group (Fig. 6).
Distribution: West Flores; only known from the type locality.
Remarks: A series of Spilarctia sp. with dark brown forewings from Timor in the collection of the Museum Witt (CMWM), München, fits well with the description of S. wahri (Rothschild, 1933), although De Vos and Suhartawan (2011: p. 313) assume that it may be an undescribed species.

Discussion
Based on the collection materials, we list eight new tiger moth species in the fauna of Flores.Among the novel records, Spilarctia mikeli sp.nov.and Aloa cardinalis danau ssp.nov.were described as local endemic taxa new to science.Together with published records, the Arctiini fauna of Flores comprises 22 species (Table 1), but this list is surely still incomplete.However, the fauna includes seven endemic taxa, with four possible local island species and three subspecies (Table 1).These findings indicate that the level of endemism in tiger moth fauna of the Flores Island (32% of putative endemic taxa) is rather moderate as compared with that in the Lasiocampidae, which contains ten species, including at least nine possible local endemics (90% of local endemic taxa) (Zolotuhin and Witt 2005).
The results of our selective DNA barcoding suggest that the tiger moth fauna of Flores includes relatively recent immigrants from Sundaland (e.g., Amerila astreus and Creatonotos gangis) and putative ancient relict elements (Spilarctia mikeli sp.nov.).Previously, divergent phylogenetic lineages, the range of which is confined to the Flores Island, were discovered in other groups of Lepidoptera, e.g., in the Lymantriinae (Bolotov et al., 2017).In summary, these results highlight that the Lesser Sunda Islands may harbor many unique species-and subspecies-level lineages of moths that are still waiting to be discovered.Broad-scale molecular studies of the Wallacean Lepidoptera remain necessary to estimate the true levels of endemism across different island archipelagoes and separate islands.

Figure 1 .
Figure 1.Map of the study region.The red ball indicates West Flores, the type locality of Spilarctia mikeli sp.nov.and Aloa cardinalis danau ssp.nov.The base of the map was modified from Lohman et al. (2011).

Figure 6 .
Figure 6.Fifty-percent majority-rule consensus phylogenetic tree of Spilarctia spp.and related taxa recovered from Bayesian analysis with 26 in-group COI haplotypes (see Table 2 for details).A haplotype of Theretra natashae was used as an out-group.Black numbers near branches are Bayesian posterior probabilities.The scale bar indicates branch lengths (nucleotide substitutions per site).

Table 1 .
List of tiger moth species recorded from Flores Island, Lesser Sunda Archipelago (Lepidoptera: Erebidae:

Table 2 .
List of COI sequences examined in this study.

Spilarctia mikeli Bolotov, Kondakov & Spitsyn sp. nov.
This new species is named in honor of Mr. Mikel Albarran Valle, an enthusiastic amateur naturalist, who lives on Flores Island.