Research Article |
Corresponding author: Yalin Zhang ( yalinzh@nwsuaf.edu.cn ) Academic editor: Carlos Peña
© 2015 Mingsheng Yang, Yalin Zhang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Yang M, Zhang Y (2015) Phylogenetic utility of ribosomal genes for reconstructing the phylogeny of five Chinese satyrine tribes (Lepidoptera, Nymphalidae). ZooKeys 488: 105-120. https://doi.org/10.3897/zookeys.488.9171
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Satyrinae is one of twelve subfamilies of the butterfly family Nymphalidae, which currently includes nine tribes. However, phylogenetic relationships among them remain largely unresolved, though different researches have been conducted based on both morphological and molecular data. However, ribosomal genes have never been used in tribe level phylogenetic analyses of Satyrinae. In this study we investigate for the first time the phylogenetic relationships among the tribes Elymniini, Amathusiini, Zetherini and Melanitini which are indicated to be a monophyletic group, and the Satyrini, using two ribosomal genes (28s rDNA and 16s rDNA) and four protein-coding genes (EF-1α, COI, COII and Cytb). We mainly aim to assess the phylogenetic informativeness of the ribosomal genes as well as clarify the relationships among different tribes. Our results show the two ribosomal genes generally have the same high phylogenetic informativeness compared with EF-1α; and we infer the 28s rDNA would show better informativeness if the 28s rDNA sequence data for each sampling taxon are obtained in this study. The placement of the monotypic genus Callarge Leech in Zetherini is confirmed for the first time based on molecular evidence. In addition, our maximum likelihood (ML) and Bayesian inference (BI) trees consistently show that the involved Satyrinae including the Amathusiini is monophyletic with high support values. Although the relationships among the five tribes are identical among ML and BI analyses and are mostly strongly-supported in BI analysis, those in ML analysis are lowly- or moderately- supported. Therefore, the relationships among the related five tribes recovered herein need further verification based on more sampling taxa.
Butterfly, molecular systematics, Bayesian inference, Satyrinae
The butterfly subfamily Satyrinae, comprising approximately 2,500 described extant species, is amongst the most diverse groups in insects (
It is widely accepted that selecting suitable genetic markers is of great importance in study of molecular systematics. In previous phylogenetic studies on the tribe level relationships of Satyrinae, the protein-coding genes (e.g., mitochondrial COI, and a number of nuclear genes) have been the main source of phylogenetic information (
In order to test the phylogenetic utility of the ribosome genes for constructing the tribe level relationships of Satyrinae which have not been resolved based on morphological and protein-coding sequence data, two ribosomal genes (16s rDNA and 28s rDNA) as well as four additional protein-coding genes (COII, Cytb, COI and EF-1α) are used in our study to reconstruct the phylogeny of the Elymniini, Amathusiini, Zetherini, Melanitini and Satyrini which represent all the major lineages of Chinese satyrines. Besides, we further clarify the taxonomic placement of the Callarge Leech, a satyrine genus which has never been included in previous molecular studies.
A total of 30 species were included in the analyses (Table
Samples used for molecular analyses in this study together with relevant information.
Subfamily | Tribe | Species | Specimen voucher | Collecting locality | GenBank accession number | |||||
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COI | COII | Cytb | 16s rDNA | EF-1α | 28s rDNA | |||||
Libytheinae | Libythea myrrha | limyr1 | China: Yunnan, Jinghong | KC158418* | KJ777775 | KJ805831 | KJ777730 | KJ805856 | KJ777756 | |
Danainae | Danaini | Danaus genutia | dagen1 | China: Yunnan, Hekou | KF226386* | KJ777776 | KJ805832 | KJ777731 | KJ805857 | KJ777757 |
Parantica sita | pasit1 | China: Yunnan, Rili | NC_024412* | KJ777777 | KJ805833 | KJ777732 | KJ805858 | KJ777758 | ||
Euploea mulciber | eumul | China: Yunnan, Lincang | NC_016720* | KJ777778 | KJ805834 | KJ777733 | KJ805859 | KJ777759 | ||
Apaturinae | Apatura ilia | apili1 | China: Hunan, Zhangjiajie | NC_016062* | KJ777779 | KJ805835 | KJ777734 | KJ805860 | KJ777760 | |
Biblidinae | Biblidini | Ariadne merione | armer1 | China: Yunnan, Lincang | KC755827* | KJ777780 | KJ805836 | KJ777735 | KJ805861 | KJ777761 |
Calinaginae | Calinaga davidis | cadav1 | China: Sichuan, Mt. Qingchengshan | NC_015480* | KJ777781 | KJ805837 | KJ777736 | KJ805862 | n.a. | |
Charaxinae | Charaxini | Charaxes bernardus | chber1 | China: Yunnan, Hekou | EF534101* | KJ777782 | KJ805838 | KJ777737 | KJ805863 | n.a. |
Polyura eudamippus | poeud1 | China: Sichuan, Pinwu | AB855881* | KJ777783 | KJ805839 | KJ777738 | KJ805864 | n.a. | ||
Satyrinae | Melanitini | Melanitis leda | meled1 | China: Yunnan, Hekou | KM111608 | KJ777784 | KJ805840 | KJ777739 | KJ805865 | KJ777762 |
Melanitis phedima | mephe1 | China: Fujian, Dehua | KM111609 | KJ777785 | KJ805841 | KJ777740 | KJ805866 | n.a. | ||
Elymniini | Elymnias hypermnestra | elhyp1 | China: Yunnan, Hekou | KM111610 | KJ777786 | KJ805842 | KJ777741 | KJ805867 | KJ777763 | |
Elymnias malelas | elmal1 | China: Xizang, Motuo | KM111611 | KJ777787 | KJ805843 | KJ777742 | KJ805868 | KJ777764 | ||
Zetherini | Callarge sagitta | casag1 | China: Gansu, Wenxian | KM111612 | KJ777788 | KJ805844 | KJ777743 | KJ805869 | KJ777765 | |
Ethope noirei | NW121-7 | Vietnam | DQ338773* | n.a. | n.a. | n.a. | DQ338915* | n.a. | ||
Penthema adelma | peade1 | China: Gansu, Wenxian | EF534103* | KJ777789 | KJ805845 | KJ777744 | KJ805870 | n.a. | ||
Penthema darlisa | CP-B02 | Vietnam | DQ338775* | n.a. | n.a. | n.a. | DQ338917* | n.a. | ||
Satyrini | Lopinga achine | loach1 | China: Shaanxi, Baoji | KM111631 | KJ777792 | KJ805848 | KJ777748 | KJ805874 | KJ777767 | |
Hipparchia autonoe | hiaut1 | China: Qinghai, Huzhu | KM111644 | KJ777794 | KJ805850 | KJ777750 | KJ805876 | KJ777769 | ||
Ninguta schrenkii | nisch1 | China: Shaanxi, Huoditang | KM111641 | KJ777793 | KJ805849 | KJ777749 | KJ805875 | KJ777768 | ||
Lethe albolineata | lealb1 | China: Yunnan, Jinghong | KM111634 | KJ777795 | KJ805851 | KJ777751 | KJ805877 | KJ777770 | ||
Tatinga tibetana | tatib1 | China: Shaanxi, Baoji | KM111633 | KJ777796 | KJ805852 | KJ777752 | KJ805878 | KJ777771 | ||
Neope pulaha | nepul1 | China: Sichuan, Pingwu | KM111640 | KJ777797 | KJ805853 | KJ777753 | KJ805879 | KJ777772 | ||
Mycalesis mamerta | mymam1 | China: Yunnan, Jinping | KM111627 | KJ777798 | KJ805854 | KJ777754 | KJ805880 | KJ777773 | ||
Minois dryas | midry1 | China: Shaanxi, Baoji | KM111645 | KJ777799 | KJ805855 | KJ777755 | KJ805881 | KJ777774 | ||
Amathusiini | Stichophthalma howqua | sthow1 | China: Yunnan, Hekou | AY218250* | KJ777790 | n.a. | KJ777745 | KJ805871 | n.a. | |
Faunis aerope | faaer1 | China: Zhejiang, Danxi | n.a. | KJ777791 | KJ805846 | KJ777746 | KJ805872 | n.a. | ||
Amathusia phidippus | NW114-17 | Indonesia | DQ018956* | n.a. | n.a. | n.a. | DQ018923* | n.a. | ||
Thauria lathyi | thlat1 | China: Yunnan, Jinghong | KM111613 | n.a. | KJ805847 | KJ777747 | KJ805873 | KJ777766 | ||
Discophora necho | NW101-6 | Indonesia | DQ338747* | n.a. | n.a. | n.a. | DQ338887* | n.a. |
Genomic DNA was extracted from 95–100% ethanol-preserved muscle tissue of two adult butterfly legs, using an EasyPure Genomic DNA Kit according to the manufacturer’s instructions (TransGen Biotech Co., Led., Beijing, China). Extracted genomic DNA was eventually dissolved in 80 µL ddH2O and kept in a freezer (–20 °C) until it was used for polymerase chain reaction (PCR). Sequences of six nuclear and mitochondrial genes (EF-1α, 28s rDNA, COI, COII, Cytb and 16s rDNA) were amplified through PCR in a total volume of 25 µL. The volume consisted of 12.5 µL CWBIO 2 × Taq MasterMix, 8.5 µL sterile distilled H2O, 2.0 µL genomic DNA template and 1.0 µL 10 µM each primer. The primers used and corresponding annealing temperature in PCR as well as references are listed in Table
Gene | Primer name (forward or reverse reading) | Sequence | Annealing temperature | References |
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COI | LCO1490 (f) | GGT CAA CAA ATC ATA AAG ATA TTG G | 51 °C |
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HCO2198 (r) | TAA ACT TCA GGG TGA CCA AAA AAT CA |
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COII | EVA (f) | GAG ACC ATT ACT TGC TTT CAG TCA CT | 53 °C |
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PATRICK (r) | CTA ATA TGG CAG ATT ATA TGT ATT GG |
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Cytb | CB-N3665 (f) | GTC CTA CCA TGA GGT CAA ATA TC | 50 °C |
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CB-N11526 (r) | TTC AAC TGG TCG TGC TCC AAT TCA |
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16s rDNA | LR-J-12887 (f) | CCG GTT TGA ACT CAG ATC ACG T | 49 °C |
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LR-N-13398 (r) | CGC CTG TTT ATC AAA AAC AT |
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EF-1α | ELF2F (f) | AAA ATG CCC TGG TTC AAG GGA | 52 °C–57 °C |
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ef51.9 (f) | CAR GAC GTA TAC AAA ATC GG |
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efrcM4 (r) | ACA GCV ACK GTY TGY CTC ATR TC |
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28s rDNA | rD3.2a (f) | AGT ACG TGA AAC CGT TCA SGG GT | 58.8 °C |
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Rd4.2b (r) | CCT TGG TCC GTG TTT CAA GAC GG |
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Sequence chromatogram was checked carefully using Chromas Pro software (Technelysium Pty Ltd., Tewntin, Australia). Each protein-coding sequence was translated for confirmation and assignment of codon positions in Primer Premier version 5.00 software (Premier Biosoft International, Palo Alto, CA). Multiple sequences were aligned using MAFFT version 7.037 with the auto strategy (
Maximum likelihood (ML) analysis was performed using the raxmlGUI version 1.3 interface (
Bayesian inference (BI) analyses were conducted in MrBayes 3.1.2 (
We used phylogenetic informativeness (PI) profiles to quantify the relative contribution of each partition to the resulted tree. The peak of the PI distribution is suggested to predict the maximum phylogenetic informativeness for corresponding partition (
One hundred and fifty-four sequences of the six genes were obtained for 30 species (Tables
COI | COII | Cytb | 16s rDNA | EF-1α | 28s rDNA | |
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Number of sequences | 29 | 25 | 25 | 26 | 30 | 19 |
Alignment length (bp) | 621 | 690 | 591 | 530 | 510 | 460 |
Percentage A(%) | 29.6 | 34.9 | 31.7 | 37.7 | 25.5 | 15.5 |
Percentage T(%) | 39.5 | 41.6 | 43.3 | 41.6 | 26.1 | 18.2 |
Percentage C(%) | 16.7 | 13.4 | 16.0 | 12.8 | 25.9 | 33.8 |
Percentage G(%) | 14.2 | 10.1 | 9.0 | 7.9 | 22.5 | 32.5 |
Number of variable sites | 233 | 288 | 275 | 167 | 165 | 184 |
Number of parsimony informative sites | 203 | 222 | 226 | 125 | 139 | 138 |
Chi-square test of base frequency | p = 1.000 | p = 1.000 | p = 0.998 | p = 1.000 | p = 0.999 | p = 0.138 |
Each gene partition shows the GTR + I + G for its best-fit substitution model except the 28s rDNA being the GTR + G, but we imposed the GTR + G for all gene partitions in ML analysis as recommended by
The best-fit partitioning schemes and corresponding partition models used in BI analysis.
Partitioned dataset | Nucleotide model under BIC | Implemented parameters in BI analysis |
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1) COI 1st + COII 1st + Cytb 1st | GTR + I + G | nst = 6, rates = invgamma |
2) COI 2nd + COII 2nd + Cytb 2nd | HKY + I + G | nst = 2, rates = invgamma |
3) COI 3rd + COII 3rd + Cytb 3rd | HKY + G | nst = 2, rates = gamma |
4) 16s rDNA | GTR + I + G | nst = 6, rates = invgamma |
5) 28s rDNA | GTR + G | nst = 6, rates = gamma |
6) EF-1α 1st | TrN + I | nst = 6, rates = inv |
7) EF-1α 2nd | JC | nst = 1 |
8) EF-1α 3rd | GTR + G | nst = 6, rates = gamma |
The ML and BI trees based on the full six-gene-dataset show generally identical topologies (summarized in Figure
The trees constructed based on the non-COI + Cytb + COII-3rds-dataset is shown in Figure
As shown in Figure
The studies of molecular systematics have been increasingly accessible because more genetic markers have been developed with the advances of sequencing technology. However, how to make informed choice to these markers confuses many systematics (
We do not recommend the use of the 3rd positions of combined COI, Cytb and COII in high level systematics of Satyrinae, although these sites show higher phylogenetic signals than other partitions (Figure
In this study, we present the first use of the ribosomal genes in reconstructing the tribe level relationships of the Satyrinae. The “satyrine” clade consisting of Calinaginae, Charaxinae and Satyrinae defined by
Among the five tribes of Satyrinae analyzed, our results recover the Satyrini as the basal lineage with a long-branch split from the rest four tribes, in agreement with the findings of
The monotypic genus Callarge is distributed restrictedly in China and on the northern border of Vietnam. Morphologically, this genus has marked black veins and lacks eyespots on wings. It is currently placed in Zetherini of Satyrinae (
We express our sincere thanks to John Richard Schrock, Emporia State University, Emporia, USA for revising the manuscript. This research is supported by the Ministry of Science and Technology of the People’s Republic of China (2011FY120200, 2006FY120100).