Corresponding author: Yalin Zhang (
Academic editor: C. Peña
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. doi:
The butterfly subfamily
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
In order to test the phylogenetic utility of the ribosome genes for constructing the tribe level relationships of
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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limyr1 | China: Yunnan, Jinghong |
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dagen1 | China: Yunnan, Hekou |
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pasit1 | China: Yunnan, Rili |
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eumul | China: Yunnan, Lincang |
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apili1 | China: Hunan, Zhangjiajie |
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armer1 | China: Yunnan, Lincang |
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cadav1 | China: Sichuan, Mt. Qingchengshan |
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n.a. | ||
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chber1 | China: Yunnan, Hekou |
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n.a. | |
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poeud1 | China: Sichuan, Pinwu |
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n.a. | |||
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meled1 | China: Yunnan, Hekou |
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mephe1 | China: Fujian, Dehua |
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n.a. | ||
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elhyp1 | China: Yunnan, Hekou |
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elmal1 | China: Xizang, Motuo |
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casag1 | China: Gansu, Wenxian |
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NW121-7 | Vietnam | n.a. | n.a. | n.a. | n.a. | ||||
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peade1 | China: Gansu, Wenxian |
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n.a. | |||
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CP-B02 | Vietnam | n.a. | n.a. | n.a. | n.a. | ||||
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loach1 | China: Shaanxi, Baoji |
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hiaut1 | China: Qinghai, Huzhu |
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nisch1 | China: Shaanxi, Huoditang |
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lealb1 | China: Yunnan, Jinghong |
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tatib1 | China: Shaanxi, Baoji |
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nepul1 | China: Sichuan, Pingwu |
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mymam1 | China: Yunnan, Jinping |
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midry1 | China: Shaanxi, Baoji |
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sthow1 | China: Yunnan, Hekou |
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n.a. |
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n.a. | ||
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faaer1 | China: Zhejiang, Danxi | n.a. |
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n.a. | ||
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NW114-17 | Indonesia | n.a. | n.a. | n.a. | n.a. | ||||
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thlat1 | China: Yunnan, Jinghong |
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n.a. |
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NW101-6 | Indonesia | n.a. | n.a. | n.a. | n.a. |
Note: * indicates the sequence downloaded from GenBank; n.a. indicates the corresponding gene fragment is not available.
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 (
Primers in PCRs for multiple genes used in this study.
Gene | Primer name (forward or reverse reading) | Sequence | Annealing temperature | References |
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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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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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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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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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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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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
Sequence statistics for the six gene regions.
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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 |
Each gene partition shows the GTR + I + G for its best-fit substitution model except the
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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GTR + I + G | nst = 6, rates = invgamma |
2) |
HKY + I + G | nst = 2, rates = invgamma |
3) |
HKY + G | nst = 2, rates = gamma |
4) |
GTR + I + G | nst = 6, rates = invgamma |
5) |
GTR + G | nst = 6, rates = gamma |
6) |
TrN + I | nst = 6, rates = inv |
7) |
JC | nst = 1 |
8) |
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-
50% majority-rule trees obtained from Bayesian inference (BI) analyses based on the non-
As shown in Figure
Phylogenetic informative profiles for all subsets used in this study.
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
In this study, we present the first use of the ribosomal genes in reconstructing the tribe level relationships of the
Among the five tribes of
The monotypic genus
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).