http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–6 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1038788
FULL LENGTH RESEARCH PAPER
DNA barcoding Satyrine butterflies (Lepidoptera: Nymphalidae) in China Mingsheng Yang1, Qing Zhai1,2, Zhaofu Yang1, and Yalin Zhang1 1
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/28/15 For personal use only.
Key Laboratory of Plant Protection Resources and Pest Management of the Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi, China and 2College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan, China Abstract
Keywords
We investigated the effectiveness of the standard 648 bp mitochondrial COI barcode region in discriminating among Satyrine species from China. A total of 214 COI sequences were obtained from 90 species, including 34 species that have never been barcoded. Analyses of genetic divergence show that the mean interspecific genetic divergence is about 16-fold higher than within species, and little overlap occurs between them. Neighbour-joining (NJ) analyses showed that 48 of the 50 species with two or more individuals, including two cases with deep intraspecific divergence (43%), are monophyletic. Furthermore, when our sequences are combined with the conspecific sequences sampled from distantly geographic regions, the ‘‘barcoding gap’’ still exists, and all related species are recovered to be monophyletic in NJ analysis. Our study demonstrates that COI barcoding is effective in discriminating among the satyrine species of China, and provides a reference library for their future molecular identification.
Biodiversity, butterfly, COI, mitochondrial DNA, molecular taxonomy
Introduction Identifying extant organisms accurately and unambiguously is of great importance in the face of current global biodiversity crisis (Guerra-Garcı´a et al., 2008). In the context of reduced taxonomic expertise in morphology, the availability of molecular data make this issue alternatively accessible (Brown et al., 1999; Tautz, 2002, 2003). Based on analysis of the partial mitochondrial cytochrome c oxidase I (COI) sequences, Hebert et al. (2003a, b) proposed an identification system to facilitate species identification and discovery for animals. The taxonomic effectiveness of this system relies on the fact that genetic variation in the COI barcode region is usually lower within than between species (i.e. barcode gap; Hebert et al., 2004b) and also those different members of a single species usually form a monophyletic clade in a barcode tree (Hebert et al., 2004a; Lukhtanov et al., 2009). Despite early opposition (Mallet & Willmot, 2003; Wiemers & Fiedler, 2007), the effectiveness of the COI-based identification system in discriminating species has now been well demonstrated in various animal groups such as birds (Hebert et al., 2004b), fishes (Hubert et al., 2008; Ward et al., 2005), bats (Clare et al., 2007) and butterflies (Ashfaq et al., 2013; Gaikwad et al., 2012; Hajibabaei et al., 2006; Hebert et al., 2004a; Janzen et al., 2005; Lukhtanov et al., 2009). The butterfly subfamily Satyrinae (Lepidoptera: Nymphalidae), includes approximately 2500 described extant species distributed around the world (Ackery et al., 1999; Pen˜a & Wahlberg, 2008). The tribe Satyrini, roughly accounting for 80% of Satyrine species, radiated rapidly between 32 and 24 Mya (Pen˜a & Wahlberg, 2008;
History Received 16 March 2015 Revised 30 March 2015 Accepted 5 April 2015 Published online 28 May 2015
Pen˜a et al., 2011). This kind of evolutionary pattern indicates that the Satyrinae is a perfect candidate for testing the ability of COI barcoding in discriminating species. However, few studies on the Satyrinae have been conducted. Although, Southeast Asia has a high level of species diversity, the number of records on the Barcode of Life Data System (BOLD, Ratnasingham & Hebert, 2007) is relatively small (Ashfaq et al., 2013). This situation is worst for China, the largest country in Southeast Asia and one spanning both the Palearctic and Oriental regions. Although, barcoding investigations including Satyrines have been conducted in some regions of Southeast Asia, such as Central Asia (Lukhtanov et al., 2009), Western India (Gaikwad et al., 2012) and Pakistan (Ashfaq et al., 2013), many species, particularly the Satyrine species occurring in China (Chou, 1999) have not been examined. In this study, we newly generated and analyzed 214 COI barcode sequences from 90 species representing approximately 25% Satyrines in China. Our objectives are to examine the effectiveness of COI barcoding in discriminating these species, and to provide a reference for future molecular identification of this group. To test further ability of COI barcoding in discriminating the species with sampling of larger geographical dimension, 54 additional barcode sequences from 16 species in previous studies (Ashfaq et al., 2013; Gaikwad et al., 2012; Lukhtanov et al., 2009) were analyzed independently, combined with our 47 sequences of these species.
Materials and methods Sampling
Correspondence: Yalin Zhang, Key Laboratory of Plant Protection Resources and Pest Management of the Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China. Tel: +86 029 8709 2190. E-mail: [email protected]
The samples examined in this study were collected during 2006– 2013 from various locations in China. Whenever possible, individuals of the same species were collected from different
2
M. Yang et al.
Mitochondrial DNA, Early Online: 1–6
places. Detailed specimen information including co-ordinates and images are available on BOLD (Ratnasingham & Hebert, 2007) in the project CNNYM (Nymphalidae (Satyrinae) of China). Identification of each individual was based on external morphology of the adult species. The majority of voucher specimens used in this study are deposited at Northwest A&F University, Yangling, China; others are from the collections of Nankai University, Tianjin, China and Hebei University, Shijiazhuang, China. All sequences generated in this study have also been submitted to GenBank, and the accession numbers can be found
in Figure 2. Additional 54 COI sequences of 16 Satyrine species downloaded from GenBank were included in our analyses (for detail information, see Table 1). DNA extraction, PCR amplification and sequencing Sequences for the standard COI barcode region newly generated in this study were obtained using two differing methods by teams at different labs. Most of the sequences were generated by the core facility at the Biodiversity Institute of Ontario,
Table 1. List of the 54 additional COI barcode sequences representing 16 satyrine species downloaded from GenBank.
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/28/15 For personal use only.
Species
Number of individual
Collecting region
Genbank number
Reference
FJ663346–FJ663348 FJ664018–FJ664020 FJ664034–FJ664036 FJ664032–FJ664033 FJ663597–FJ663602 FJ663267–FJ663269 FJ663767–FJ663768 FJ663646–FJ663648 FJ663379–FJ663382 FJ663395–FJ663396 FJ663733 GU012553–GU012554, GU012602, GU012619, GU012624 GU012586, GU012598 KC158411 KC158412–KC158414, HQ990376 KC158407–KC158410, HQ990368–HQ990372
Lukhtanov et al. (2009)
Chazara briseis Pseudochazara hippolyte Satyrus ferula Minois dryas Hipparchia autonoe Aphantopus hyperanthus Melanargia russiae Hyponephele lupina Coenonympha amaryllis Coenonympha oedippus Lopinga achine Melanitis leda
3 3 3 2 6 4 2 3 4 2 1 5
Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Western Ghats of India
Orsotriaena medus Lethe rohria Lethe verma Lasiommata schakra
2 1 4 9
Western Ghats of India Pakistan Pakistan Pakistan
Gaikwad et al. (2012) Ashfaq et al. (2013)
Figure 1. Distributions of intraspecific and interspecific genetic divergences based on 214 mitochondrial COI sequences for 90 species of Chinese Satyrines.
DNA barcoding Satyrine butterflies (Lepidoptera: Nymphalidae) in China
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DOI: 10.3109/19401736.2015.1038788
Figure 2. Neighbour-joining tree (including the A, B and C parts) based on the 214 COI sequences of 90 satyrine species from China. Bootstrap values 550 are not shown; the species marked with red bar on the right have not been barcoded previously.
University of Guelph using methods similar to those described in Yang et al. (2012). Other sequences were obtained at Northwest A&F University of China, employing the following procedures. Genomic DNA was extracted from two dried or 95–100% ethanol-preserved legs using an Easy Pure Genomic DNA Kit (TransGen Biotech Co., Ltd., Beijing, China). The target 658 bp fragment of COI was amplified through polymerase chain reaction (PCR) using the standard barcoding primer pair LCO1490 (50 -GGT CAA CAA ATC ATA AAG ATA TTG G30 ) and HCO2198 (50 -TAA ACT TCA GGG TGA CCA AAA AAT CA-30 ) (Folmer et al., 1994). The following thermal profile was used for 25 mL amplification reactions: initial denaturation for 5 min at 94 C, followed by 34 cycles of 0.5 min at 94 C, 0.5 min at 51 C, 1 min at 72 C and a subsequent final extension at 72 C
for 7 min. After the PCR products were checked with 1% agarose gel, sequencing was performed at Sunny Biotechnology Co., Ltd. (Shanghai, China) using the same primers as in PCR. Data analysis Multiple sequence alignments were conducted with ClustalX version 2.02 (Larkin et al., 2007), and then sequences were edited manually with MEGA version 6.06 (Tamura et al., 2013). The average genetic divergences within and between species were calculated using the Kimura 2-Parameter (K2P) model (Kimura, 1980). The same model was used to construct the NeighborJoining (NJ) tree at 1000 pseudoreplicates (Saitou & Nei, 1987). Both K2P genetic divergences and NJ tree were generated using MEGA version 6.06 (Tamura et al., 2013).
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Mitochondrial DNA, Early Online: 1–6
Figure 3. Distributions of intraspecific and interspecific genetic divergences based on 101 mitochondrial COI sequences of 16 satyrine species.
Results A total of 214 specimens representing 90 species belonging to 37 genera of Chinese Satyrines were analyzed. Among these individuals, two or more individuals were included for 50 species (56% of the 90 species). The 50 fragment of the COI mitochondrial gene was amplified and sequenced bi-directionally for each individual, and the lengths were from 540 to 658 bp (658 bp for about 74% of the 214 sequences). The partial COI sequences from 90 species had high A + T content (69.1% on average), particularly in the third codon position (89.4%). Intraspecific genetic divergences ranged from 0.0 to 5.1%, with an average of 0.8%. High intraspecific genetic divergences (43%) were detected in two species: Lopinga achine and Neope muirheadii. Interspecific genetic divergence ranged from 1.1% to 19.6%, with an average of 12.9%. Low levels of interspecific genetic divergence (52%) typically occurred between closely related congeneric species, i.e. Melanargia epimede and M. halimede, Aphantopus hyperantus and A. arvensis, and Chonala episcopalis and C. praeusta. Overall, the interspecific mean genetic divergence was about 16-fold higher than intraspecific divergence, with little overlap between intraspecific and interspecific divergence values (Figure 1). The NJ tree (Figure 2) showed that most species represented by two or more individuals were recovered as monophyletic with high bootstrap values (495%). Two exceptions were Elymnias malelas and M. epimede. Although L. achine and N. muirheadii had relatively high intraspecific genetic divergences, they were also recovered as monophyletic by NJ analysis. The combined analysis of the 101 sequences from 16 species showed that the intraspecific genetic divergences for 14 of the 16 species were under 2%, with an average of 1.4%. The other two species and corresponding intraspecific genetic divergences were Lopinga achine (3.5%) and Hyponephele lupina (3.6%). Interspecific genetic divergence ranged from 8.2% to 16.7%,
with an average of 12.7%. All species showed no overlapping between intraspecific and interspecific divergence values (Figure 3). The NJ tree (Figure 4) showed that all species were recovered as monophyletic with high bootstrap values (495%).
Discussion We present the first investigation on DNA barcodes of Chinese Satyrines with 214 COI sequences from 90 species. Moreover, until this study, 34 (as indicated in Figure 2) of these species lacked barcode records on BOLD (Ratnasingham & Hebert, 2007) and GenBank. This should be helpful for future molecular identification of these species, particularly for the 34 species never studied previously. The existence of a ‘‘barcoding gap’’ and reciprocal monophyly of the 96% (excluding E. malelas and M. epimede) of 50 species represented by two or more individuals indicate that this COIbased identification system is effective in discriminating the Satyrine species examined in this study. The average intraspecific genetic divergence was 0.8%, higher than noted in previous studies (0.17% in Hajibabaei et al., 2006; 0.26% in Gaikwad et al., 2012; and 0.2% in Ashfaq et al., 2013) on butterflies. This high value (0.8%) is definitely related to the two species (Lopinga achine and Neope muirheadii) with unusual high intraspecific genetic divergences (43%). Interestingly, our samples of the former species include representatives of two subspecies (L. a. achinoides, L. a. catena). As these two subspecies are distantly allopatric, we suspect that they may actually represent separate species, but this needs to be investigated further. Similarly, the four specimens of N. muirheadii included one from Southwest China (N. m. felderi) and three from Southeast China representing a different subspecies (N. m. muirheadii). Surprisingly, one individual from Southeast China showed high divergence (7.6%) than the other two sympatric individuals, but low divergence (0.3%) than subspecies from Southwest China. Therefore, we
DNA barcoding Satyrine butterflies (Lepidoptera: Nymphalidae) in China
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DOI: 10.3109/19401736.2015.1038788
Figure 4. Neighbour-joining tree (including the A and B parts) based on the 101 COI sequences of 16 satyrine species. Bootstrap values 550 are not shown.
suspect this specimen may be wrongly labeled. Furthermore, the deep divergence between the two subspecies also indicates that further taxonomic investigations are needed for N. muirheadii. For Lepidoptera, Hebert et al. (2003a) found that the genetic divergences between species are generally greater than 3%. Our results support this finding, with 99% of the species pairs showing a genetic divergence greater than this value. The three exceptions with an extremely low divergence (1–1.6%) are Melanargia epimede and M. halimede, Aphantopus hyperantus and A. arvensis, and Chonala episcopalis and C. praeusta. The two closely related species of Melanargia belong to the same subgenus Halimede (Nazari et al., 2010; Oberthu¨r & Houlbert, 1922; Verity, 1953). The low divergence between them is supported by Nazari et al. (2010) that shows the un-corrected
p distance as 1.29 ± 0.26%. Besides, Pen˜a et al. (2011) suggested that the Melanargiina (including the Melanargia only) is the youngest subtribe in the Satyrini. Therefore, we infer that the two species of the Melanargia perhaps as well as the Aphantopus and Chonala are recently diverged. In addition to measures of genetic divergence, NJ analysis is also effective in assessing the utility of COI barcoding in discriminating animal species and has been widely used in prior barcoding studies (Ashfaq et al., 2013; Gaikwad et al., 2012; Lukhtanov et al., 2009). Although, conspecific individuals from distant geographic places often show more genetic divergence, Lukhtanov et al. (2009) reported that they remain monophyletic in NJ analyses. This conclusion is supported by our study as both L. achine and N. muirheadii were recovered as monophyletic,
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although the sampled individuals derived from distant geographic areas and showed deep intraspecific divergences. Based on combining data of 16 species examined in this study and previous studies (Ashfaq et al., 2013; Gaikwad et al., 2012; Lukhtanov et al., 2009), the intraspecific divergences are increased in the following species compared with that used data only generated in this study: Chazara briseis, Melanargia russiae, Melanitis leda, Coenonympha oedippus, Orsotriaena medus and A. hyperanthus. However, conspecific sequences of them still form monophyletic clades. This further reinforces the previous findings (e.g. Lukhtanov et al., 2009; Gaikwad et al., 2012; Ashfaq et al., 2013) that NJ analysis is effective in discriminating species with large-scale sampling coverage based COI barcode data.
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Conclusions Overall, our study shows that COI barcoding is effective in discriminating the Satyrine species of China examined in this study. It will also be great helpful for assessing genetic diversity and population structure over large-scale geographic ranges for this most diverse group in butterfly. However, as the present study only provides COI barcodes for about 25% of Chinese Satyrines, more barcode sequences should be obtained to supplement the current barcode database for the Satyrinae of China.
Acknowledgements We express our sincere thanks to Paul Hebert (Biodiversity Institute of Ontario, University of Guelph, Guelph, Canada) for DNA sequencing and revisionary suggestions to our manuscript.
Declaration of interest This research was supported by the Ministry of Science and Technology of the People’s Republic of China (2011FY120200, 2006FY120100) and the National Natural Science Foundation of China (Grant No. 31201733). All authors have read and agree with the contents of the manuscript. The authors report no conflict of interest, including financial interests, relationships and affiliations.
References Ackery PR, de Jong R, Vane-Wright RI. (1999). The butterflies: Hedyloidea, Hesperioidea and Papilionoidea. In: Kristensen NP, editor. Lepidoptera, Moths and Butterflies. Vol. 1: Evolution, systematics, and biogeography. Handbook of zoology. Berlin: de Gruyter. p 263–300. Ashfaq M, Akhtar S, Khan AM, Adamowicz SJ, Hebert PDN. (2013). DNA barcode analysis of butterfly species from Pakistan points towards regional endemism. Mol Ecol Resour 13:832–43. Brown B, Emberson RM, Paterson AM. (1999). Mitochondrial COI and II provide useful markers for Weiseana (Lepidoptera, Hepialidae) species identification. Bull Entomol Res 89:287–94. Chou I. (1999). Monograph of Chinese butterflies, First Volume (Revised edition). Zhengzhou, China: Henan Scientific and Technological Publishing House (in Chinese with English Abstract). Clare EL, Lim BK, Engstrom MD, Eger JL, Hebert PDN. (2007). DNA barcoding of Neotropical bats: Species identification and discovery within Guyana. Mol Ecol Notes 7:184–190. Folmer O, Black M, Hoeh W, Lutz R. Vrijenhoek R. (1994). DNA primers for amplification of mitochondrial cytochrome oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3: 294–9. Gaikwad SS, Ghate HV, Ghaskadbi SS, Patole MS, Shouche YS. (2012). DNA barcoding of nymphalid butterflies (Nymphalidae: Lepidoptera) from Western Ghats of India. Mol Biol Reports 39:2375–83.
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Guerra-Garcı´a JM, Espinosa F, Garcı´a-Go´mez JC. (2008). Trends in taxonomy today: An overview about the main topics in Taxonomy. Zoologica Baetica 19:15–49. Hajibabaei M, Janzen DH, Burns JM, Hallwachs W, Hebert PDN. (2006). DNA barcodes distinguish species of tropical Lepidoptera. Proc Natl Acad Sci USA 103:968–71. Hebert PDN, Cywinska A, Ball SL, DeWaard JR. (2003a). Biological identifications through DNA barcodes. Proc Royal Soc B 270:313–21. Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W. (2004a). Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA 101:14812–17. Hebert PDN, Ratnasingham S, deWaard JR. (2003b). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Royal Soc B 270:S96–9. Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM. (2004b). Identification of birds through DNA barcodes. PLoS Biol 2:e312. Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M, Watkinson D, et al. (2008). Identifying Canadian freshwater fishes through DNA barcodes. PLoS ONE 3:e2490. Janzen DH, Hajibabaei M, Burns JM. (2005). Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding. Philos Trans R Soc Lond B Biol Sci 1462:1835–46. Kimura M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–20. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–8. Lukhtanov VA, Sourakov A, Zakharov EV, Hebert PDN. (2009). DNA barcoding central Asian butterfly: Increasing geographical dimension does not significantly reduce the success of species identification. Mol Ecol Resour 9:1302–10. Mallet J, Willmot K. (2003). Taxonomy: Renaissance or tower of babel? Trends Ecol Evol 18:57–9. Nazari V, Hagen WT, Bozano GC. (2010). Molecular systematics and phylogeny of the ‘Marbled Whites’ (Lepidoptera: Nymphalidae, Satyrinae, Melanargia Meigen). Syst Entomol 35:132–47. Oberthu¨r C, Houlbert C. (1922). Quelques vues nouvelles sur la systematique des Melanargia (Lepidopteres: Satyridae). Comptes Rendus de l’Academie des Sciences Paris 174:190–2. Pen˜a C, Wahlberg N. (2008). Prehistorical climate change increased diversification of a group of butterflies. Biol Lett 4:274–8. Pen˜a C, Nylin S, Wahlberg N. (2011). The radiation of Satyrini butterflies (Nymphalidae: Satyrinae): A challenge for phylogenetic methods. Zool J Linn Soc 161:64–87. Ratnasingham S, Hebert PDN. (2007). BOLD: the Barcode of life data system. Mol Ecol Notes 7:355–64. Saitou N, Nei M. (1987). The neighbour-joining method: A new method for reconstructing evolutionary trees. Mol Biol Evol 4:406–25. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–9. Tautz D, Arctander P, Minelli A, Thomas RH, Vogler AP. (2002). DNA points the way ahead in taxonomy. Nature 41:479. Tautz D, Arctander P, Minelli A, Thomas RH, Vogler AP. (2003). A plea for DNA taxonomy. Trends Ecol Evol 18:70–4. Verity R. (1953). Le Farfalle Diurne d’italia. Volume Quinto. Divisione Papilionida. Sezione Nymphalina: Famiglia Satyridae. Florence, Italy: Casa editrice Marzocco. Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN. (2005). DNA barcoding Australia’s fish species. Philos Transac Royal Soc Lon Ser B Biol Sci 360:1847–57. Wiemers M, Fiedler K. (2007). Does the DNA barcoding gap exist? – a case study in blue butterflies (Lepidoptera: Lycaenidae). Front Zool 4:8–23. Yang Z, Landry J-F, Zhang Y, Solis MA, Handfield D, Scholtens BG, Mutanen M, et al. (2012). DNA barcoding and morphology reveal three cryptic species of Anania (Lepidoptera: Crambidae: Pyraustinae) in North America, all distinct from their European counterpart. Syst Entomol 37:686–705.
FULL LENGTH RESEARCH PAPER
DNA barcoding Satyrine butterflies (Lepidoptera: Nymphalidae) in China Mingsheng Yang1, Qing Zhai1,2, Zhaofu Yang1, and Yalin Zhang1 1
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/28/15 For personal use only.
Key Laboratory of Plant Protection Resources and Pest Management of the Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi, China and 2College of Plant Protection, Henan Agricultural University, Zhengzhou, Henan, China Abstract
Keywords
We investigated the effectiveness of the standard 648 bp mitochondrial COI barcode region in discriminating among Satyrine species from China. A total of 214 COI sequences were obtained from 90 species, including 34 species that have never been barcoded. Analyses of genetic divergence show that the mean interspecific genetic divergence is about 16-fold higher than within species, and little overlap occurs between them. Neighbour-joining (NJ) analyses showed that 48 of the 50 species with two or more individuals, including two cases with deep intraspecific divergence (43%), are monophyletic. Furthermore, when our sequences are combined with the conspecific sequences sampled from distantly geographic regions, the ‘‘barcoding gap’’ still exists, and all related species are recovered to be monophyletic in NJ analysis. Our study demonstrates that COI barcoding is effective in discriminating among the satyrine species of China, and provides a reference library for their future molecular identification.
Biodiversity, butterfly, COI, mitochondrial DNA, molecular taxonomy
Introduction Identifying extant organisms accurately and unambiguously is of great importance in the face of current global biodiversity crisis (Guerra-Garcı´a et al., 2008). In the context of reduced taxonomic expertise in morphology, the availability of molecular data make this issue alternatively accessible (Brown et al., 1999; Tautz, 2002, 2003). Based on analysis of the partial mitochondrial cytochrome c oxidase I (COI) sequences, Hebert et al. (2003a, b) proposed an identification system to facilitate species identification and discovery for animals. The taxonomic effectiveness of this system relies on the fact that genetic variation in the COI barcode region is usually lower within than between species (i.e. barcode gap; Hebert et al., 2004b) and also those different members of a single species usually form a monophyletic clade in a barcode tree (Hebert et al., 2004a; Lukhtanov et al., 2009). Despite early opposition (Mallet & Willmot, 2003; Wiemers & Fiedler, 2007), the effectiveness of the COI-based identification system in discriminating species has now been well demonstrated in various animal groups such as birds (Hebert et al., 2004b), fishes (Hubert et al., 2008; Ward et al., 2005), bats (Clare et al., 2007) and butterflies (Ashfaq et al., 2013; Gaikwad et al., 2012; Hajibabaei et al., 2006; Hebert et al., 2004a; Janzen et al., 2005; Lukhtanov et al., 2009). The butterfly subfamily Satyrinae (Lepidoptera: Nymphalidae), includes approximately 2500 described extant species distributed around the world (Ackery et al., 1999; Pen˜a & Wahlberg, 2008). The tribe Satyrini, roughly accounting for 80% of Satyrine species, radiated rapidly between 32 and 24 Mya (Pen˜a & Wahlberg, 2008;
History Received 16 March 2015 Revised 30 March 2015 Accepted 5 April 2015 Published online 28 May 2015
Pen˜a et al., 2011). This kind of evolutionary pattern indicates that the Satyrinae is a perfect candidate for testing the ability of COI barcoding in discriminating species. However, few studies on the Satyrinae have been conducted. Although, Southeast Asia has a high level of species diversity, the number of records on the Barcode of Life Data System (BOLD, Ratnasingham & Hebert, 2007) is relatively small (Ashfaq et al., 2013). This situation is worst for China, the largest country in Southeast Asia and one spanning both the Palearctic and Oriental regions. Although, barcoding investigations including Satyrines have been conducted in some regions of Southeast Asia, such as Central Asia (Lukhtanov et al., 2009), Western India (Gaikwad et al., 2012) and Pakistan (Ashfaq et al., 2013), many species, particularly the Satyrine species occurring in China (Chou, 1999) have not been examined. In this study, we newly generated and analyzed 214 COI barcode sequences from 90 species representing approximately 25% Satyrines in China. Our objectives are to examine the effectiveness of COI barcoding in discriminating these species, and to provide a reference for future molecular identification of this group. To test further ability of COI barcoding in discriminating the species with sampling of larger geographical dimension, 54 additional barcode sequences from 16 species in previous studies (Ashfaq et al., 2013; Gaikwad et al., 2012; Lukhtanov et al., 2009) were analyzed independently, combined with our 47 sequences of these species.
Materials and methods Sampling
Correspondence: Yalin Zhang, Key Laboratory of Plant Protection Resources and Pest Management of the Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China. Tel: +86 029 8709 2190. E-mail: [email protected]
The samples examined in this study were collected during 2006– 2013 from various locations in China. Whenever possible, individuals of the same species were collected from different
2
M. Yang et al.
Mitochondrial DNA, Early Online: 1–6
places. Detailed specimen information including co-ordinates and images are available on BOLD (Ratnasingham & Hebert, 2007) in the project CNNYM (Nymphalidae (Satyrinae) of China). Identification of each individual was based on external morphology of the adult species. The majority of voucher specimens used in this study are deposited at Northwest A&F University, Yangling, China; others are from the collections of Nankai University, Tianjin, China and Hebei University, Shijiazhuang, China. All sequences generated in this study have also been submitted to GenBank, and the accession numbers can be found
in Figure 2. Additional 54 COI sequences of 16 Satyrine species downloaded from GenBank were included in our analyses (for detail information, see Table 1). DNA extraction, PCR amplification and sequencing Sequences for the standard COI barcode region newly generated in this study were obtained using two differing methods by teams at different labs. Most of the sequences were generated by the core facility at the Biodiversity Institute of Ontario,
Table 1. List of the 54 additional COI barcode sequences representing 16 satyrine species downloaded from GenBank.
Mitochondrial DNA Downloaded from informahealthcare.com by Nyu Medical Center on 05/28/15 For personal use only.
Species
Number of individual
Collecting region
Genbank number
Reference
FJ663346–FJ663348 FJ664018–FJ664020 FJ664034–FJ664036 FJ664032–FJ664033 FJ663597–FJ663602 FJ663267–FJ663269 FJ663767–FJ663768 FJ663646–FJ663648 FJ663379–FJ663382 FJ663395–FJ663396 FJ663733 GU012553–GU012554, GU012602, GU012619, GU012624 GU012586, GU012598 KC158411 KC158412–KC158414, HQ990376 KC158407–KC158410, HQ990368–HQ990372
Lukhtanov et al. (2009)
Chazara briseis Pseudochazara hippolyte Satyrus ferula Minois dryas Hipparchia autonoe Aphantopus hyperanthus Melanargia russiae Hyponephele lupina Coenonympha amaryllis Coenonympha oedippus Lopinga achine Melanitis leda
3 3 3 2 6 4 2 3 4 2 1 5
Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Central Asian Western Ghats of India
Orsotriaena medus Lethe rohria Lethe verma Lasiommata schakra
2 1 4 9
Western Ghats of India Pakistan Pakistan Pakistan
Gaikwad et al. (2012) Ashfaq et al. (2013)
Figure 1. Distributions of intraspecific and interspecific genetic divergences based on 214 mitochondrial COI sequences for 90 species of Chinese Satyrines.
DNA barcoding Satyrine butterflies (Lepidoptera: Nymphalidae) in China
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DOI: 10.3109/19401736.2015.1038788
Figure 2. Neighbour-joining tree (including the A, B and C parts) based on the 214 COI sequences of 90 satyrine species from China. Bootstrap values 550 are not shown; the species marked with red bar on the right have not been barcoded previously.
University of Guelph using methods similar to those described in Yang et al. (2012). Other sequences were obtained at Northwest A&F University of China, employing the following procedures. Genomic DNA was extracted from two dried or 95–100% ethanol-preserved legs using an Easy Pure Genomic DNA Kit (TransGen Biotech Co., Ltd., Beijing, China). The target 658 bp fragment of COI was amplified through polymerase chain reaction (PCR) using the standard barcoding primer pair LCO1490 (50 -GGT CAA CAA ATC ATA AAG ATA TTG G30 ) and HCO2198 (50 -TAA ACT TCA GGG TGA CCA AAA AAT CA-30 ) (Folmer et al., 1994). The following thermal profile was used for 25 mL amplification reactions: initial denaturation for 5 min at 94 C, followed by 34 cycles of 0.5 min at 94 C, 0.5 min at 51 C, 1 min at 72 C and a subsequent final extension at 72 C
for 7 min. After the PCR products were checked with 1% agarose gel, sequencing was performed at Sunny Biotechnology Co., Ltd. (Shanghai, China) using the same primers as in PCR. Data analysis Multiple sequence alignments were conducted with ClustalX version 2.02 (Larkin et al., 2007), and then sequences were edited manually with MEGA version 6.06 (Tamura et al., 2013). The average genetic divergences within and between species were calculated using the Kimura 2-Parameter (K2P) model (Kimura, 1980). The same model was used to construct the NeighborJoining (NJ) tree at 1000 pseudoreplicates (Saitou & Nei, 1987). Both K2P genetic divergences and NJ tree were generated using MEGA version 6.06 (Tamura et al., 2013).
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Figure 3. Distributions of intraspecific and interspecific genetic divergences based on 101 mitochondrial COI sequences of 16 satyrine species.
Results A total of 214 specimens representing 90 species belonging to 37 genera of Chinese Satyrines were analyzed. Among these individuals, two or more individuals were included for 50 species (56% of the 90 species). The 50 fragment of the COI mitochondrial gene was amplified and sequenced bi-directionally for each individual, and the lengths were from 540 to 658 bp (658 bp for about 74% of the 214 sequences). The partial COI sequences from 90 species had high A + T content (69.1% on average), particularly in the third codon position (89.4%). Intraspecific genetic divergences ranged from 0.0 to 5.1%, with an average of 0.8%. High intraspecific genetic divergences (43%) were detected in two species: Lopinga achine and Neope muirheadii. Interspecific genetic divergence ranged from 1.1% to 19.6%, with an average of 12.9%. Low levels of interspecific genetic divergence (52%) typically occurred between closely related congeneric species, i.e. Melanargia epimede and M. halimede, Aphantopus hyperantus and A. arvensis, and Chonala episcopalis and C. praeusta. Overall, the interspecific mean genetic divergence was about 16-fold higher than intraspecific divergence, with little overlap between intraspecific and interspecific divergence values (Figure 1). The NJ tree (Figure 2) showed that most species represented by two or more individuals were recovered as monophyletic with high bootstrap values (495%). Two exceptions were Elymnias malelas and M. epimede. Although L. achine and N. muirheadii had relatively high intraspecific genetic divergences, they were also recovered as monophyletic by NJ analysis. The combined analysis of the 101 sequences from 16 species showed that the intraspecific genetic divergences for 14 of the 16 species were under 2%, with an average of 1.4%. The other two species and corresponding intraspecific genetic divergences were Lopinga achine (3.5%) and Hyponephele lupina (3.6%). Interspecific genetic divergence ranged from 8.2% to 16.7%,
with an average of 12.7%. All species showed no overlapping between intraspecific and interspecific divergence values (Figure 3). The NJ tree (Figure 4) showed that all species were recovered as monophyletic with high bootstrap values (495%).
Discussion We present the first investigation on DNA barcodes of Chinese Satyrines with 214 COI sequences from 90 species. Moreover, until this study, 34 (as indicated in Figure 2) of these species lacked barcode records on BOLD (Ratnasingham & Hebert, 2007) and GenBank. This should be helpful for future molecular identification of these species, particularly for the 34 species never studied previously. The existence of a ‘‘barcoding gap’’ and reciprocal monophyly of the 96% (excluding E. malelas and M. epimede) of 50 species represented by two or more individuals indicate that this COIbased identification system is effective in discriminating the Satyrine species examined in this study. The average intraspecific genetic divergence was 0.8%, higher than noted in previous studies (0.17% in Hajibabaei et al., 2006; 0.26% in Gaikwad et al., 2012; and 0.2% in Ashfaq et al., 2013) on butterflies. This high value (0.8%) is definitely related to the two species (Lopinga achine and Neope muirheadii) with unusual high intraspecific genetic divergences (43%). Interestingly, our samples of the former species include representatives of two subspecies (L. a. achinoides, L. a. catena). As these two subspecies are distantly allopatric, we suspect that they may actually represent separate species, but this needs to be investigated further. Similarly, the four specimens of N. muirheadii included one from Southwest China (N. m. felderi) and three from Southeast China representing a different subspecies (N. m. muirheadii). Surprisingly, one individual from Southeast China showed high divergence (7.6%) than the other two sympatric individuals, but low divergence (0.3%) than subspecies from Southwest China. Therefore, we
DNA barcoding Satyrine butterflies (Lepidoptera: Nymphalidae) in China
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DOI: 10.3109/19401736.2015.1038788
Figure 4. Neighbour-joining tree (including the A and B parts) based on the 101 COI sequences of 16 satyrine species. Bootstrap values 550 are not shown.
suspect this specimen may be wrongly labeled. Furthermore, the deep divergence between the two subspecies also indicates that further taxonomic investigations are needed for N. muirheadii. For Lepidoptera, Hebert et al. (2003a) found that the genetic divergences between species are generally greater than 3%. Our results support this finding, with 99% of the species pairs showing a genetic divergence greater than this value. The three exceptions with an extremely low divergence (1–1.6%) are Melanargia epimede and M. halimede, Aphantopus hyperantus and A. arvensis, and Chonala episcopalis and C. praeusta. The two closely related species of Melanargia belong to the same subgenus Halimede (Nazari et al., 2010; Oberthu¨r & Houlbert, 1922; Verity, 1953). The low divergence between them is supported by Nazari et al. (2010) that shows the un-corrected
p distance as 1.29 ± 0.26%. Besides, Pen˜a et al. (2011) suggested that the Melanargiina (including the Melanargia only) is the youngest subtribe in the Satyrini. Therefore, we infer that the two species of the Melanargia perhaps as well as the Aphantopus and Chonala are recently diverged. In addition to measures of genetic divergence, NJ analysis is also effective in assessing the utility of COI barcoding in discriminating animal species and has been widely used in prior barcoding studies (Ashfaq et al., 2013; Gaikwad et al., 2012; Lukhtanov et al., 2009). Although, conspecific individuals from distant geographic places often show more genetic divergence, Lukhtanov et al. (2009) reported that they remain monophyletic in NJ analyses. This conclusion is supported by our study as both L. achine and N. muirheadii were recovered as monophyletic,
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although the sampled individuals derived from distant geographic areas and showed deep intraspecific divergences. Based on combining data of 16 species examined in this study and previous studies (Ashfaq et al., 2013; Gaikwad et al., 2012; Lukhtanov et al., 2009), the intraspecific divergences are increased in the following species compared with that used data only generated in this study: Chazara briseis, Melanargia russiae, Melanitis leda, Coenonympha oedippus, Orsotriaena medus and A. hyperanthus. However, conspecific sequences of them still form monophyletic clades. This further reinforces the previous findings (e.g. Lukhtanov et al., 2009; Gaikwad et al., 2012; Ashfaq et al., 2013) that NJ analysis is effective in discriminating species with large-scale sampling coverage based COI barcode data.
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Conclusions Overall, our study shows that COI barcoding is effective in discriminating the Satyrine species of China examined in this study. It will also be great helpful for assessing genetic diversity and population structure over large-scale geographic ranges for this most diverse group in butterfly. However, as the present study only provides COI barcodes for about 25% of Chinese Satyrines, more barcode sequences should be obtained to supplement the current barcode database for the Satyrinae of China.
Acknowledgements We express our sincere thanks to Paul Hebert (Biodiversity Institute of Ontario, University of Guelph, Guelph, Canada) for DNA sequencing and revisionary suggestions to our manuscript.
Declaration of interest This research was supported by the Ministry of Science and Technology of the People’s Republic of China (2011FY120200, 2006FY120100) and the National Natural Science Foundation of China (Grant No. 31201733). All authors have read and agree with the contents of the manuscript. The authors report no conflict of interest, including financial interests, relationships and affiliations.
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