New records of geometrid moths ( Lepidoptera : Geometridae ) from Myanmar based on DNA barcodes and morphological data

An integrative study of the Indo-Burmese Geometridae in the collection of the Russian Museum of Biodiversity Hotspots (RMBH, Russia) revealed six newly recorded species for the fauna of Myanmar, i.e., Plutodes costatus (Butler, 1886), Pogonopygia nigralbata Warren, 1894, Mixochlora vittata (Moore, 1867), Pelagodes bellula Han & Xue, 2011, Agathia carissima Butler, 1878 and A. codina Swinhoe, 1892. The occurrence of P. bellula is the first record of this taxon outside the Tibetan Plateau. Our findings highlight that the distribution of several Oriental geometrids could be broader than was previously suggested.


Introduction
The Indo-Burma Region represents one of the world's greatest biodiversity hotspots (Martens 2015).However, the Indo-Burmese fauna of Lepidoptera is poorly known, particularly concerning large, complex taxonomy such as Geometridae.There are some works covering the geometrid fauna of certain regions such as northern India (Hampson 1895), China (Han and Xue 2011a) and Borneo (Holloway 1993(Holloway , 1996)), but there is a lack of recent publications on the fauna of Myanmar.Notwithstanding, in a book by Hampson (1895) which is mainly focused on geometrid fauna of India, there are some data in a poorly detailed study of Myanmar geometrid moths.
In available sources, the distributions of certain species are often described using vague locations, for example, the Himalayas (Holloway 1993, 1996, Cerny and Pinratana 2009, Bucsek 2012), which is actually the world's greatest mountain system that is bordered by a number of countries, including China, India, Nepal, Bhutan, and others.Such an approach can sometimes cause uncertainty during the compiling of the national checklists of Lepidoptera fauna.Accordingly, this uncertainty in the actual distribution range of taxa makes the environmental protection activity a much more complicated task.Many examples of Asian Lepidoptera species have large gaps of areas over entire countries in their distribution ranges,
In the present study, we report new distribution records of six geometrid species that are recorded from Myanmar for the first time.To confirm these new records, we performed DNA barcoding for 10 specimens, which were initially separated based on the morphological investigation.

Methods
Morphological studies.The Indo-Burmese Geometridae were studied in the collection of the Russian Museum of Biodiversity Hotspots (RMBH hereafter), Institute of Biogeography and Genetic Resources, Federal Center for Integrated Arctic Research, Russian Academy of Sciences, Arkhangelsk, Russia.The sampling localities are listed in Table 1.The specimens were prepared according to a standard protocol (Schauff 2001).The genitalia dissections were performed using common methods for Lepidoptera (Robinson 1976).The abdomen was macerated in hot 10% KOH solution for 3-5 minutes.The extracted genitalia were mounted in permanent slides with Histofluid ® (Paul Marienfeld GmbH & Co., Germany).Specimens were photographed using a digital camera (Canon EOS 650D, Canon, Japan).Genitalia slides were photographed using a stereo microscope (Ste-REO Discovery.V8, Carl Zeiss, Germany).DNA barcoding.Total DNA was extracted from a single leg of each dry specimen according to a standard phenol/chloroform procedure (Sambrook et al. 1989).The mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified and sequenced using primers C1-J-1718 (forward primer: 5ʹ GGAGGATTTGGAAATT-GATTAGTTCC 3ʹ) and C1-N-2329 (reverse primer: 5ʹ ACTGTAAATATATGATGAGCTCA 3ʹ) (Simon et al. 1994).The PCR mix contained approximately 200 ng of total cell DNA, 10 pmol of each primer, 200 μmol of each dNTP, 2.5 μl of PCR buffer (with 20 mmol MgCl 2 ), 0.8 units Taq DNA polymerase (SibEnzyme Ltd., Novosibirsk, Russia), and H 2 O was added for a final volume of 25 μl.Temperature cycling was as follows: 95 °C (4 min), 40 cycles of 95 °C (50 sec), 48 °C (50 sec), 72 °C (50 sec) and a final extension at 72 °C (5 min).Forward and reverse sequence reactions were performed directly on purified PCR products using the ABI PRISM® Big-Dye™ Terminator v. 3.1 reagents kit and run on an ABI PRISM® 3730 DNA (Thermo Fisher Scientific Inc., Waltham, MA, USA).The resulting sequences were checked using a sequence alignment editor (BioEdit version 7.2.5, Hall 1999).
All new sequences were uploaded to the NCBI Gen-Bank database (Table 2).The taxonomic affinities of our nucleotide sequences were identified using the Basic Local Alignment Search Tool, BLAST (Johnson et al. 2008) and the Barcode of Life Data Systems (BOLD Systems v3; see Ratnasingham and Hebert 2007).
Identification.The moth specimens were identified with appropriate keys (Han and Xue 2011a, 2011b, Holloway 1993, 1996) based on the external diagnostic features and male genitalia morphology.Dr H.-X. Han (Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences) confirmed our identification of Pelagodes bellula from Myanmar.

Results
In this study 10 new COI nucleotide sequences of six geometrid species were obtained (Table 2).As aforementioned, each of the six species is recorded in Myanmar for the first time.Additionally, we used a dataset of 84 barcode sequence records of these taxa that was obtained via the BOLD database.

Subfamily Ennominae
Plutodes costatus (Butler, 1886) Diagnosis.This species is similar to P. thorbeni Stüning, 2010, an endemic of Sulawesi, but differs by narrower yellow edging of the forewing and longer saccular process in the male genitalia.
DNA barcoding.With respect to the BOLD records, specimens from Myanmar show the closest affinity to a specimen from Bhutan with genetic distance of 0.22% (Table 2).In general, 26 nearest neighbor sequences with distances up to 0.95% were recorded using the BOLD database.The nearest neighbors were collected from Bhutan, Thailand, Myanmar, China, Vietnam, Malaysia and Indonesia.Diagnosis.This species is similar to numerous congeners but differs by marking patterns of the hindwing.However, DNA barcoding is the best way for its reliable identification.
DNA barcoding.With respect to the BOLD records, specimens from Myanmar revealed the closest affinity to specimens from China and Vietnam with genetic distance of 0.22% (Table 2).In general, 34 nearest neighbor sequences with distances up to 1.89% were recorded using the BOLD database.The nearest neighbors were collected from Vietnam, China, Japan, Malaysia, and Indonesia.
Remarks.Chu (1981: fig. 865) illustrated this species but erroneously identified it as Dilophodes elegans sinica, although these two taxa have clear differences in forewing morphology.

DNA barcoding.
With respect to the BOLD records, a specimen from Myanmar reveals the closest affinity to a specimen from Thailand with genetic distance of 0.22% (Table 2).In general, 10 nearest neighbor sequences with distances up to 2.46% were recorded using the BOLD database.The nearest neighbors were collected from Thailand, Nepal, Bhutan, Philippines, Malaysia, and Indonesia.
Remarks.Holloway (1996) illustrated the male genitalia of Mixochlora vittata as those of M. argentifusa and vice versa, likely because of a typographic error.Actually, the genitalia of the former species are given in Holloway's fig.169, while the genitalia of M. argentifusa are shown in figure 168 (Holloway 1996).Diagnosis.Pelagodes bellula shows great similarity with P. aucta, another species from India.In this case, the only reliable way to do a correct species identification is to compare rate of sclerotization in the male genitalia: P. bellula has lesser sclerotization compared with P. aucta (Han and Xue 2011b).
DNA barcoding.With respect to the BOLD records, specimens from Myanmar show the closest affinity to a specimen of Thalassodes veraria (this species is currently placed within Pelagodes: see Han and Xue 2011b) from Vietnam with genetic distance of 0.45% (Table 2).No other nearest neighbors were recorded.
Remarks.Our new record marks the occurrence of Pelagodes bellula outside of the territory of China for the first time (Fig. 21).We suggest that the species may also range in Vietnam.However, since the discussed specimen from the BOLD Systems dataset was identified as Thalassodes veraria, this question needs further clarification.the specimen from Myanmar reveals the closest affinity to the specimen from Vietnam with genetic distance of 0.67% (Table 2).Six additional nearest neighbor sequences with distances up to 1.57% were recorded from Thailand, Malaysia, and Indonesia.

Discussion
An integrative study of the Indo-Burmese Geometridae in the RMBH collection revealed six newly recorded species for the fauna of Myanmar.Among them, five species are common and widespread (i.e., Plutodes costatus, Pogonopygia nigralbata, Mixochlora vittata, Agathia carissima and Agathia codina).By contrast, Pelagodes bellula is a recently described species, which was known by four type specimens collected near Yarang (29.296°N, 095.279°E) in southern Tibet (Han and Xue 2011b).The distance between the Tibetan locality and our collection point in Kachin is roughly 280 km (Fig. 21).The elevations of the species localities in Tibet are much greater than those in Kachin (790-1090 m and 420 m, respectively).With respect to our data, the range of P. bellula is much broader than was previously assumed and this species may also be found in northern India (e.g., in Arunachal Pradesh).
The documentation of moths and reporting their distribution with specific localities will help the ecologists to formulate the conservation plans.We presume that our study, which applies an integrative taxonomic approach to report new distributional records, will encourage new discoveries.

Table 1 .
Sampling sites in Kachin state, Myanmar

Table 2 .
List of the mitochondrial COI sequences of Geometridae obtained in the present study