Corresponding author: Haechul Park (
Academic editor: A. Frolov
These authors contributed equally to this work.
The seven species belonging to the genus
Lee G-E, Han T, Jeong J, Kim S-H, Park IG, Park H (2015) Molecular phylogeny of the genus
Genus
Among the seven species of
The subspecies of
During a review of the genus
Fifty specimens of
The male habitus of species and subspecies of
Collection and voucher information for specimens.
Sample no. | Species | Locality | Data collected | Sex | Voucher no. | Sequencing | |
---|---|---|---|---|---|---|---|
GBAn of COI | GBAn of 16S | ||||||
1 |
|
Muju, JB, South Korea | 6. VI. 2012 | F | 7258 |
|
|
2 |
|
Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea | 19. V. 2009 | M | 7300 |
|
|
3 |
|
Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea | 19. V. 2009 | M | 7301 |
|
|
4 |
|
Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea | 19. V. 2009 | M | 7302 |
|
|
5 |
|
Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea | 19. V. 2009 | F | 7303 |
|
|
6 |
|
Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea | 25. V. 2013 | M | 7696 |
|
|
7 |
|
Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea | 25. V. 2013 | M | 7697 |
|
|
8 |
|
Tongrim, North Korea | VII. 1995 | M | 7683 |
|
– |
9 |
|
North Korea | IV. 2002 | M | 7684 |
|
|
10 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7264 |
|
|
11 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7265 |
|
|
12 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7267 |
|
|
13 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7268 |
|
|
14 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7269 |
|
|
15 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7270 |
|
|
16 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7272 |
|
|
17 |
|
Mt. Wu Long, Dandong, Liaoning, China | 15. VII. 2009 | M | 7273 |
|
|
18 |
|
Sichuan, China | VI. 2008 | M | 7677 |
|
|
19 |
|
Sichuan, China | VI. 2008 | F | 7678 |
|
|
20 |
|
Sichuan, China | VI. 2008 | F | 7679 |
|
– |
21 |
|
Sichuan, China | VI. 2008 | F | 7680 |
|
|
22 |
|
Mt. Foding, Guizhou, China | – | F | 7688 |
|
|
23 |
|
Tibet, China | – | M | 7685 |
|
– |
24 |
|
Tibet, China | – | M | 7686 |
|
|
25 |
|
Tibet, China | – | F | 7687 |
|
– |
26 |
|
Tibet, China | VIII. 2005 | F | 7689 |
|
|
27 |
|
A- Li-Shan, Chiayi county, Taiwan | IV. 2012 | F | 7290 |
|
|
28 |
|
A- Li-Shan, Chiayi county, Taiwan | IV. 2012 | F | 7291 |
|
|
29 |
|
A- Li-Shan, Chiayi county, Taiwan | IV. 2012 | F | 7292 |
|
|
30 |
|
Hanyan, Sichuan, China | 16–17. VI. 2007 | M | 7278 |
|
|
31 |
|
Hanyan, Sichuan, China | 16–17. VI. 2007 | M | 7279 |
|
|
32 |
|
H-1601m, Env. Xichang city, S. Sichuan, China | 12. VI. 2009 | M | 7375 |
|
|
33 |
|
H-1601m, Env. Xichang city, S. Sichuan, China | 12. VI. 2009 | F | 7376 |
|
|
34 |
|
China | 2005 | M | 7690 |
|
|
35 |
|
Chiayi, Taiwan | VIII. 2011 | M | 7285 |
|
|
36 |
|
Chiayi, Taiwan | VIII. 2011 | M | 7286 |
|
|
37 |
|
A- Li-Shan, Chiayi county, Taiwan | IV. 2012 | M | 7287 |
|
|
38 |
|
A- Li-Shan, Chiayi county, Taiwan | IV. 2012 | M | 7288 |
|
|
39 |
|
A- Li-Shan, Chiayi county, Taiwan | IV. 2012 | M | 7289 |
|
|
40 |
|
Mt. Lianyuan, Hunan, China | VII. 2006 | M | 7692 |
|
|
41 |
|
Mt. Lianyuan, Hunan, China | VII. 2006 | F | 7693 |
|
|
42 |
|
Mt. Guangwu, Sichuan, China | – | M | 7694 |
|
|
43 |
|
Mt. Guangwu, Sichuan, China | – | F | 7695 |
|
|
44 |
|
Taeng, Mae, Mai, Ching, N. Thailand | VII. 2010 | M | 7274 |
|
|
45 |
|
Taeng, Mae, Mai, Ching, N. Thailand | IV. 2008 | M | 7275 |
|
|
46 |
|
Beitou, Taipei, Taiwan | V. 2008 | F | 7277 |
|
|
47 |
|
Beitou, Taipei, Taiwan | V. 2008 | M | 7280 |
|
|
48 |
|
Beitou, Taipei, Taiwan | V. 2008 | M | 7281 |
|
|
49 |
|
Beitou, Taipei, Taiwan | V. 2008 | F | 7282 | 198 bp |
|
50 |
|
Beitou, Taipei, Taiwan | V. 2008 | F | 7283 |
|
|
51 |
|
Korea | – | – | – |
|
|
denotes outgroup taxa data extracted from GenBank. GBAn is denoted the GenBank accession number.
Genomic DNA (gDNA) was extracted from middle legs removed from dried specimens of all species and accomplished using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Polymerase Chain Reaction (PCR) was performed in order to amplify the cytochrome
For the phylogenetic analyses, three data sets were used, a 658 bp fragment of
ML analysis was performed with GARLI 2.0 (
BI analysis was performed with MrBayes 3.1.2 (
MP analysis was performed with TNT 1.1 (
The data set of
Phylogenetic inferences based on three analyses (ML, BI, and MP) reconstructed the same topologies for COI (Fig.
Phylogenetic relationships among
The intra-specific distances of COI were rather low, ranging from 0–2.3%. The inter-specific divergences were highly variable, ranging from 2.7%–16.7%. The distances between the ingroup and outgroup taxa ranged from 16.1%–20.1% (Table
Pairwise distance of COI within and between
No. of samples | Within species | Between subspecies & species | |||||||
---|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|
||||
26 | 0.006 (0–0.017) | ||||||||
|
3 | 0.011 (0.002–0.017) | 0.062 (0.056–0.073) | ||||||
|
5 | 0.008 (0–0.015) | 0.150 (0.130–0.162) | 0.140 (0.130–0.149) | |||||
|
5 | 0.013 (0.002–0.023) | 0.150 (0.131–0.167) | 0.135 (0.128–0.150) | 0.069 (0.056–0.089) | ||||
|
4 | 0.006 (0.003–0.008) | 0.120 (0.104–0.131) | 0.117 (0.105–0.127) | 0.139 (0.130–0.152) | 0.117 (0.105–0.134) | |||
|
2 | 0.006 (0.006–0.006) | 0.133 (0.126–0.141) | 0.123 (0.121–0.124) | 0.132 (0.125–0.137) | 0.135 (0.125–0.144) | 0.048 (0.043–0.050) | ||
|
5 | 0.003 (0–0.006) | 0.123 (0.109–0.134) | 0.122 (0.120–0.124) | 0.146 (0.131–0.163) | 0.128 (0.104–0.147) | 0.060 (0.048–0.081) | 0.047 (0.027–0.057) | |
|
1 | – | 0.175 (0.168–0.179) | 0.168 (0.164–0.170) | 0.196 (0.192–0.201) | 0.191 (0.188–0.196) | 0.179 (0.166–0.188) | 0.198 (0.197–0.199) | 0.176 (0.161–0.189) |
Numbers are indicated as mean (minimum-maximum) of the pairwise distance.
denotes outgroup taxon
Clade A is composed of
Clade B is composed of
ML, BI, and MP analyses of 16S rRNA resulted in considerably similar topologies to those of COI (Fig.
Phylogenetic relationships among
The intra-specific pairwise distances of
Pairwise distance of 16S ribosomal RNA within and between
No. of samples | Within species | Between subspecies & species | |||||||
---|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|
||||
22 | 0.000 (0.000–0.002) | ||||||||
|
3 | 0.001 (0.000–0.002) | 0.009 (0.008–0.012) | ||||||
|
5 | 0.002 (0.000–0.004) | 0.057 (0.054–0.060) | 0.050 (0.046–0.052) | |||||
|
5 | 0.001 (0.000–0.002) | 0.059 (0.058–0.063) | 0.052 (0.050–0.054) | 0.020 (0.018–0.022) | ||||
|
4 | 0.001 (0.000–0.003) | 0.046 (0.042–0.055) | 0.039 (0.034–0.049) | 0.035 (0.028–0.047) | 0.036 (0.032–0.047) | |||
|
2 | 0.000 (0.000–0.000) | 0.050 (0.050–0.050) | 0.043 (0.042–0.044) | 0.030 (0.030–0.032) | 0.034 (0.034–0.036) | 0.009 (0.008–0.011) | ||
|
5 | 0.001 (0.000–0.002) | 0.048 (0.048–0.048) | 0.041 (0.040–0.042) | 0.032 (0.028–0.034) | 0.034 (0.032–0.036) | 0.012 (0.008–0.016) | 0.015 (0.014–0.016) | |
|
1 | – | 0.104 (0.104–0.106) | 0.102 (0.101–0.104) | 0.103 (0.101–0.104) | 0.104 (0.104–0.104) | 0.103 (0.097–0.118) | 0.099 (0.099–0.099) | 0.101 (0.099–0.102) |
Numbers are indicated as mean (minimum-maximum) of the pairwise distance.
denotes outgroup taxon
In the combined data set of COI and
Phylogenetic relationships among
The 19 diagnostic characters used to classify species or subspecies were re-examined in order to determine whether they are suitable for identification (Table
Anterior edge of clypeus of
Apicosutural angle of
Umbone (in the circle) of shoulder of
Diagnostic characters of
Character | states | Reference | |
---|---|---|---|
Body | 1. Color in male (Fig. |
0) grayish brown |
|
1) dark brown | |||
2) yellowish brown | |||
3) dark yellowish brown | |||
4) green-yellowish brown with pale purple on elytra | |||
2. Color in female | 0) dark blackish body without marking |
|
|
1) not dark blackish body | |||
3. Pronotal and elytral colors (Fig. |
0) pronotum and elytra different |
|
|
1) pronotum and elytra similar | |||
4. Dorsal surface | 0) pilose with brownish semirecumbent hairs |
|
|
1) almost hairless | |||
2) sparsely pilose with hair | |||
Head | 5. Development of antlers | 0) a pair of antlers in male very short, undeveloped, approximate to each other anteriorly |
|
1) antlers in male long and well developed, curving upwards apically and broadly separated from each other | |||
6. Inferior dentation of antlers | 0) clearly projected upward |
|
|
1) weakly prominent | |||
2) absent | |||
7. Shape of anterior edge of clypeus (Fig. |
0) simple without angular projection |
|
|
1) with an angular projection | |||
8. Circular indentation of clypeus | 0) with a strong or weak circular indentation on the edge |
|
|
1) without circular indentation on the edge | |||
Pronotum | 9. Pronotal bands | 0) reaching posterior border |
|
1) not reaching posterior border | |||
10. Central carinae | 0) carinae defined |
|
|
1) carinae nearly indistinct | |||
11. Extending of carinae | 0) extending beyond the middle |
|
|
1) never extending beyond the middle | |||
2) no carina | |||
12. The widest portion | 0) widest near the middle |
|
|
1) widest in front of the middle | |||
Elytra | 13. Surface | 0) with two black dots |
|
1) without black dot | |||
14. Shoulder (Fig. |
0) with triangular umbone |
|
|
1) without triangular umbone | |||
15. Apicosutural angle (Fig. |
0) rounded |
|
|
1) projected | |||
Metasternum | 16. Metasternal process | 0) obtuse, rather rounded |
|
1) rectangular or acute, moderately produced | |||
2) triangularly and sharply produced | |||
Abdomen | 17. Abdominal sternites in male | 0) covered with yellowish grey powder |
|
1) normal, not covered with yellowish grey powder | |||
Legs | 18. Color of tarsi | 0) clear reddish brown (=testaceous) |
|
1) black or very dark brown | |||
19. Length of tarsi | 0) anterior tarsi of the male about as long as posterior ones |
|
|
1) anterior tarsi distinctly longer than the others |
Data matrix for
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
0 |
|
|
|
? | 1 | 0 | 1 | 1 |
|
|
|
1 (rarely 0) | 0 |
|
0 | 1 |
|
|
|
|
|
|
|
? |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0 |
|
1 | 0 |
|
|
|
|
|
|
0 (or 1) |
|
|
? | 1 |
|
|
1 |
|
|
1 | 0 (or 1) | 1 |
|
? | 1 | 1 | 0 |
|
|
|
0 |
|
0 | 0 | 1 | 1 |
|
|
1 |
|
|
0 | 0 |
|
|
|
|
|
|
|
|
|
? | 0 |
|
|
0 |
|
|
1 | 1 |
|
|
0 (or 1) | 0 (or 1) | 1 | 1 | 0 | 0 |
|
|
0 | 0 |
|
1 | 0 |
|
|
|
1 | 1 |
|
|
|
0 (or 1) | 1 | 1 | 0 | 0 |
|
|
|
0 | 2 | 1 | 0 |
|
|
4 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
|
1 | 1 (rarely 0) |
|
|
1 | 1 | 1 | 0 | 1 |
Results of examination | C | C | U | C | C | U | U | C | C | C | C | U | C | U | U | C | C | C | C |
Boldic numbers indicate additionally examined diagnostic characters at each species in this study.
Parentheses denote the characteristic represeted by our examination.
Question marks indicate the ambiguous character state to be difficult determination in our examination.
‘C’ is clear and ‘U’ is unclear characters resulted in this study.
Metasternal process (in the circle) and aedeagi of
From the results inferred from ML, BI, and MP methods using COI and 16S rRNA genes, the genus
In all topologies,
In contrast with the molecular data of the
Regarding the status of the subspecies of
The three subspecies of
We are very grateful to Dr. David Mark Boshko of Editing Korea, Seoul, Korea, Mr. Aaron Butt of University of South Alabama, Mobile, USA, for the English revision of the manuscript and two anonymous reviewers for providing valuable suggestions and comments that improved this work. This study was carried out with the support of the Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ00939502), Rural Development Administration, Republic of Korea
(DNA sequences)
This COI data includes 50 individual sequences of the examined
(DNA sequences)
This 16S rRNA data includes 46 individual sequences of the examined
The combined dataset of COI and 16S rRNA of
(DNA sequences)
There is the concatenated sequences of COI and 16S rRNA genes correspondence with each sample.