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Research Article
Molecular phylogeny of the genus Dicronocephalus (Coleoptera, Scarabaeidae, Cetoniinae) based on mtCOI and 16S rRNA genes
expand article infoGa-Eun Lee, Taeman Han, Jongchel Jeong§, Seong-Hyun Kim, In Gyun Park, Haechul Park
‡ National Academy of Agricultural Science, Jeonju, Korea, South
§ Seodaemun Museum of Natural History, Seoul, Korea, South
Open Access

Abstract

The seven species belonging to the genus Dicronocephalus are a very interesting group with a unique appearance and distinct sexual dimorphism. Only one species among them, D. adamsi, has been known in the Korean fauna. This species is recognized as having a wide distribution from Tibet to Korean Peninsula and is currently represented by two subspecies that have separated geographical ranges. The phylogenetic relationships of D. adamsi were still unclear. The phylogeny of Dicronocephalus is reconstructed with a phylogenetic study of five species including four subspecies based on a molecular approach using mitochondrial COI and 16S rRNA genes. Our results are compared with the results obtained by previous authors based on morphological characters. They show that the tested taxa are divided into two major clades. Clade A consists of two species (D. adamsi + D. yui) and Clade B includes the others (D. dabryi + D. uenoi + D. wallichii). This result generally supports Kurosawa’s proposal except that D. dabryi and D. uenoi are newly recognized as members of a monophyletic group. We propose that D. adamsi drumonti is a junior subjective synonym of D. adamsi adamsi. These results show that three members of the D. wallichii group should be treated as species rather than subspecies. However, further research including analyses of different genetic markers is needed to reconfirm our results.

Keywords

Dicronocephalus, phylogenetic relationships, D. adamsi, taxonomy, Scarabaeidae, new synonymy, Korea

Introduction

Genus Dicronocephalus Hope, 1831 is a group of medium- to large-sized beetles with a unique appearance among Cetoniinae representatives. The members of the genus show distinct sexual dimorphism such as antler-like clypeal horns and prolonged tarsomeres in males (Šípek et al. 2008). This genus is composed of seven species including nine subspecies: D. adamsi adamsi Pascoe, 1863; D. adamsi drumonti Legrand, 2005; D. dabryi (Lucas, 1872); D. shimomurai Kurosawa, 1986; D. uenoi uenoi Kurosawa, 1968; D. uenoi katoi Kurosawa, 1968; D. bieti Pouillaude, 1914; D. wallichii wallichii Hope, 1831; D. wallichii bourgoini Pouillaude, 1914; D. wallichii bowringi Pascoe, 1863; D. yui yui Kurosawa, 1968; and D. yui cheni Kurosawa, 1986 (Legrand 2005, Krajcik 2014). Geographically, the genus is widely distributed from the Himalayan foothills of Nepal to Vladivostok in Russia and to Korea, but the distribution of most species and subspecies is rather limited. In particular, D. shimomurai, D. uenoi uenoi, D. uenoi katoi, D. wallichii bourgoini, D. yui yui, and D. yui cheni are endemic to the small island of Taiwan. One species, D. dabryi, is only known in West China and Myanmar. The remaining species and subspecies are widely distributed in Asia occurring throughout the Manchuria and Indo-China (Kurosawa 1986, Šípek et al. 2008, Young 2012, Krajcik 2014).

Kurosawa (1986) proposed dividing this genus into three groups on the basis of the morphological characters: 1) the adamsi species-group (D. adamsi, D. shimomurai, and D. yui); 2) the wallichii species-group (D. w. wallichii, D. w. bourgoini, D. w. bowringi, and D. dabryi); and 3) the D. uenoi species-group (D. uenoi). However, he did not explain the phylogenetic relationships between these species.

Among the seven species of Dicronocephalus, only D. adamsi is found in the Korean fauna. This species was described from Korea, but it has been known to have a wide range across Korea, China, Tibet, and Vietnam. The range of this species is divided by a wide geographical gap between Liaoning and Shanxi provinces of China (Young 2012). Legrand (2005) divided D. adamsi into two subspecies based on this distribution pattern and morphological differences. He described populations occurring in west China as D. adamsi drumonti. This classification was accepted by Krajcik (2014), but not by Young (2012).

The subspecies of D. wallichii (D. w. wallichii, D. w. bourgoini, and D. w. bowringi) were originally described as valid species (Hope 1831, Pascoe 1863, Pouillaude 1914). While some authors have treated these taxa as subspecies (Paulian 1960, Mikšić 1971, 1977, Krajčík 1998, Sakai and Nagai 1998, Šípek et al. 2008, Young 2012, Krajcik 2014), some others have treated them as species (Kurosawa 1968, Devecis 2008). The controversy over whether they should be dealt with at the species or sub-species level has continued without in-depth analysis.

During a review of the genus Dicronocephalus, several issues were encountered, such as validation of species or subspecies rank of taxa composing D. adamsi and D. wallichi (sensu lato) and the lack of phylogenetic analysis of the genus. To resolve these questions, phylogenetic analysis was performed for the genus using cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA) mitochondrial gene sequences as well as examination of their morphological diagnostic characters.

Materials and methods

Specimen sampling and examination

Fifty specimens of Dicronocephalus belonging to five species and seven subspecies from four countries were obtained (Fig. 1, Table 1), but we were unable to obtain specimens of the remaining two species, D. bieti and D. shimomurai. For examining male genitalia, these were extracted from the abdomens and cleaned by heating with 10% KOH solution in a WiseTherm®HB-48P heating block at 60 °C for 1~2 hours. Male genitalia were preserved in microvials with glycerine after examination. Photographs of external morphology and genitalia were taken with a Canon EOS 10D camera and stacked with a combineZM program (Hadley 2006). Based on previous studies (Pascoe 1863, Pouillaude 1914, Kurosawa 1968, 1986, Young 2012), diagnostic characters were obtained to provide precise criteria for species identification. In this study, the most recent taxonomic scheme by Krajcik (2014) was followed, especially for subspecies treatment of D. wallichii. All examined specimens are stored in the Department of Agricultural Biology, National Academy of Agricultural Biology (NAAS), Jeonju, Korea.

Figure 1.

The male habitus of species and subspecies of Dicoronocephalus. A D. adamsi adamsi B D. a. drumonti C D. yui yui D D. dabryi E D. uenoi katoi F D. wallichii bowringi G D. w. wallichii H D. w. bourgoini.

Collection and voucher information for specimens.

Sample no. Species Locality Data collected Sex Voucher no. Sequencing
GBAn of COI GBAn of 16S
1 Dicronocephalus adamsi adamsi Muju, JB, South Korea 6. VI. 2012 F 7258 KM390855 KM390809
2 Dicronocephalus adamsi adamsi Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea 19. V. 2009 M 7300 KM390856 KM390810
3 Dicronocephalus adamsi adamsi Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea 19. V. 2009 M 7301 KM390857 KM390811
4 Dicronocephalus adamsi adamsi Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea 19. V. 2009 M 7302 KM390858 KM390812
5 Dicronocephalus adamsi adamsi Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea 19. V. 2009 F 7303 KM390859 KM390813
6 Dicronocephalus adamsi adamsi Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea 25. V. 2013 M 7696 KM390860 KM390814
7 Dicronocephalus adamsi adamsi Sangdaewon-dong, Jungwon-gu, Seongnam, GG, South Korea 25. V. 2013 M 7697 KM390861 KM390815
8 Dicronocephalus adamsi adamsi Tongrim, North Korea VII. 1995 M 7683 KM390862
9 Dicronocephalus adamsi adamsi North Korea IV. 2002 M 7684 KM390863 KM390816
10 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7264 KM390864 KM390817
11 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7265 KM390865 KM390818
12 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7267 KM390866 KM390819
13 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7268 KM390867 KM390820
14 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7269 KM390868 KM390821
15 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7270 KM390869 KM390822
16 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7272 KM390870 KM390823
17 Dicronocephalus adamsi adamsi Mt. Wu Long, Dandong, Liaoning, China 15. VII. 2009 M 7273 KM390871 KM390824
18 Dicronocephalus adamsi drumonti Sichuan, China VI. 2008 M 7677 KM390872 KM390825
19 Dicronocephalus adamsi drumonti Sichuan, China VI. 2008 F 7678 KM390873 KM390826
20 Dicronocephalus adamsi drumonti Sichuan, China VI. 2008 F 7679 KM390874
21 Dicronocephalus adamsi drumonti Sichuan, China VI. 2008 F 7680 KM390875 KM390827
22 Dicronocephalus adamsi drumonti Mt. Foding, Guizhou, China F 7688 KM390876 KM390828
23 Dicronocephalus adamsi drumonti Tibet, China M 7685 KM390877
24 Dicronocephalus adamsi drumonti Tibet, China M 7686 KM390878 KM390829
25 Dicronocephalus adamsi drumonti Tibet, China F 7687 KM390879
26 Dicronocephalus adamsi drumonti Tibet, China VIII. 2005 F 7689 KM390880 KM390830
27 Dicronocephalus yui yui A- Li-Shan, Chiayi county, Taiwan IV. 2012 F 7290 KM390881 KM390831
28 Dicronocephalus yui yui A- Li-Shan, Chiayi county, Taiwan IV. 2012 F 7291 KM390882 KM390832
29 Dicronocephalus yui yui A- Li-Shan, Chiayi county, Taiwan IV. 2012 F 7292 KM390883 KM390833
30 Dicronocephalus dabryi Hanyan, Sichuan, China 16–17. VI. 2007 M 7278 KM390884 KM390834
31 Dicronocephalus dabryi Hanyan, Sichuan, China 16–17. VI. 2007 M 7279 KM390885 KM390835
32 Dicronocephalus dabryi H-1601m, Env. Xichang city, S. Sichuan, China 12. VI. 2009 M 7375 KM390886 KM390836
33 Dicronocephalus dabryi H-1601m, Env. Xichang city, S. Sichuan, China 12. VI. 2009 F 7376 KM390887 KM390837
34 Dicronocephalus dabryi China 2005 M 7690 KM390888 KM390838
35 Dicronocephalus uenoi katoi Chiayi, Taiwan VIII. 2011 M 7285 KM390889 KM390839
36 Dicronocephalus uenoi katoi Chiayi, Taiwan VIII. 2011 M 7286 KM390890 KM390840
37 Dicronocephalus uenoi katoi A- Li-Shan, Chiayi county, Taiwan IV. 2012 M 7287 KM390891 KM390841
38 Dicronocephalus uenoi katoi A- Li-Shan, Chiayi county, Taiwan IV. 2012 M 7288 KM390892 KM390842
39 Dicronocephalus uenoi katoi A- Li-Shan, Chiayi county, Taiwan IV. 2012 M 7289 KM390893 KM390843
40 Dicronocephalus wallichii bowringi Mt. Lianyuan, Hunan, China VII. 2006 M 7692 KM390894 KM390844
41 Dicronocephalus wallichii bowringi Mt. Lianyuan, Hunan, China VII. 2006 F 7693 KM390895 KM390845
42 Dicronocephalus wallichii bowringi Mt. Guangwu, Sichuan, China M 7694 KM390896 KM390846
43 Dicronocephalus wallichii bowringi Mt. Guangwu, Sichuan, China F 7695 KM390897 KM390847
44 Dicronocephalus wallichii wallichii Taeng, Mae, Mai, Ching, N. Thailand VII. 2010 M 7274 KM390898 KM390848
45 Dicronocephalus wallichii wallichii Taeng, Mae, Mai, Ching, N. Thailand IV. 2008 M 7275 KM390899 KM390849
46 Dicronocephalus wallichii bourgoini Beitou, Taipei, Taiwan V. 2008 F 7277 KM390900 KM390850
47 Dicronocephalus wallichii bourgoini Beitou, Taipei, Taiwan V. 2008 M 7280 KM390901 KM390851
48 Dicronocephalus wallichii bourgoini Beitou, Taipei, Taiwan V. 2008 M 7281 KM390902 KM390852
49 Dicronocephalus wallichii bourgoini Beitou, Taipei, Taiwan V. 2008 F 7282 198 bp KM390853
50 Dicronocephalus wallichii bourgoini Beitou, Taipei, Taiwan V. 2008 F 7283 KM390903 KM390854
51 Protaetia brevitarsis* Korea KC775706 KC775706

DNA extraction, amplification and sequencing

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 c oxidase subunit I gene (COI) and 16S ribosomal RNA gene (16S rRNA) using Accupower PCR PreMix (Bioneer, Daejeon, Korea). The universal primer set LCO1490/HCO2198 (Folmer et al. 1994) for amplifying the DNA barcoding region (658bp) of COI sequences was not successful for all samples; this may be caused by the degraded quality of gDNA (Goldstein and Desalle 2003, Hajibabaei et al. 2006; Wandeler et al. 2007). We applied the PCR methodology for retrieving COI sequences from old specimens given in Han et al. (2014) and designed new primer pairs: LCO-Ceto232F (5’–GCHTTYCCYCGAATAAATAAYATA–3’) corresponding to HCO2198 and HCO-Ceto367R (5’–ACDGTYCADCCNGTTCCTGCNCC–3’) corresponding to LCO1490. 16S rRNA was targeted in a 600 bp region with two primers, 16SB/16SA, that successfully amplified in Lucanidae and Elateridae (Hosoya et al. 2001, Hosoya and Araya 2005, Han et al. 2009, 2010). PCR amplification conditions were as follows: for COI, initial denaturation at 94 °C for 5 min, then 45 cycles at 94 °C for 30 s, 46 °C for 25 s, and 72 °C for 45 s followed by a final extension at 72 °C for 3 min, and for 16S rRNA, initial denaturation at 94 °C for 5 min, then 40 cycles at 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 45 s followed by a final extension at 72 °C for 5min. The amplicons were purified using a QIA quick PCR Purification Kit (Qiagen, Hilden, Germany) after the product yield was monitored by 0.7% agarose gel electrophoresis. DNA sequencing was performed using an automated DNA sequencer (ABI 3730xl 96-capillary DNA analyzer; Applied Biosystems, Foster City, CA) with the same primers used for PCR. All sequences (excepting a 198 bp fragment of COI in no. 7282) are available from GenBank under accession numbers KM390855KM390903 for COI and KM390809KM390854 for 16S rRNA (Table 1).

Phylogenetic analysis

For the phylogenetic analyses, three data sets were used, a 658 bp fragment of COI, 520 bp fragment of 16S rRNA sequences, and the concatenated COI and 16S rRNA sequences. The data sets were aligned using ClustalW in MEGA 5.2 (Tamura et al. 2011), and genetic distances were calculated using Kimura’s two-parameter test (Kimura 1980). The phylogenetic analyses were constructed using maximum likelihood (ML), Bayesian inference methods (BI), and maximum parsimony (MP).

ML analysis was performed with GARLI 2.0 (Zwickl 2011), and the analysis was initiated at a random start tree using GTR+I+G model parameters selected by MrModelTest (Nylander 2004), with a 10,000 generation search algorithm and 1,000 bootstrap replications. The frequencies with which to log the best score (“logevery”) and to save the best tree to file (“saveevery”) were set to 10,000 and 10,000 respectively, and the number of generations without topology improvement required for termination (“genthreshfortopoterm”) was set to 5,000. At the end of the analysis, there was no improvement in the tree topology by a log likelihood of 0.01 or better. The bootstrap values were calculated using the SumTrees program of the DendroPy package (Sukumaran and Holder 2010).

BI analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Metropolis-coupled Markov chain Monte Carlo (MCMC) analyses were run with one cold and three heated chains (temperature set to 0.2) for 5,000,000 generations and tree sampling every 100 generations. The posterior probabilities were then obtained and a majority-rule consensus tree was generated from the remaining trees after discarding the first 25% of samples.

MP analysis was performed with TNT 1.1 (Goloboff et al. 2008). The analyses, followed by tree bisection reconnection (TBR) branch swapping, used default options that performed 100 random additional sequences and saved up to ten trees per replication. To obtain the strict consensus tree, symmetric resampling (Goloboff et al. 2003) with a 33% change probability and jack-knifing with a 36% removal probability were implemented using a traditional search with 1,000 replications. Each set of results was summarized in terms of absolute frequency, and the group support values were analyzed. For bootstrap value (BP) in ML and MP, and posterior probability value (PP) in BI, supporting values of <70% as “weak”, 70–79% as “moderate”, 80–89% as “strong”, and ≥ 90% as “very strong” support were used.

Results

Nucleotide information for COI and 16S rRNA

The data set of COI, with no evidence of indel (insertion/deletion) events, had 144 (21.9%) variable sites (Vs). Of these, 140 (21.3%) were parsimoniously informative sites (PIs). The data set of 16S rRNA, with indel events at three sites, consisted of 43 (8.3%) Vs, of which 41 (7.9%) were PIs. There was about 2.6 times more variability and the level of PIs was about 2.7 times greater in COI than in that in 16S rRNA.

Phylogenetic analyses of COI

Phylogenetic inferences based on three analyses (ML, BI, and MP) reconstructed the same topologies for COI (Fig. 2; for BI, ML and MP tree data not shown, see Suppl. material 1 for sequences), and there was separation into two major clades (A and B) with very strong supporting values (100%), except for ML. Eight ingroup taxa representatives including subspecies were clearly clustered into seven monophyletic groups corresponding to nominal species; the two subspecies of D. adamsi formed one cluster. Their terminal nodes were well supported, but the values of ML and BI were very low in D. yui yui (<50% in ML and 53% in BI) and D. wallichii bowringi (<50% in ML and 56% in BI).

Figure 2.

Phylogenetic relationships among Dicronocephalus species reconstructed with Bayesian inference using COI sequences. Numbers above branches indicate ML bootstrap values and Bayesian posterior probabilities. Numbers below branches are bootstrap, symmetric resampling, and jacknife support from parsimony searches, respectively. Scale bar represents 10% nucleotide mutation rate.

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 2).

Pairwise distance of COI within and between Dicronocephalus spp.

No. of samples Within species Between subspecies & species
D. a. adamsi + D. a. drumonti D. yui yui D. dabryi D. uenoi katoi D. w. bowringi D. w. wallichii D. w. bourgoini
D. adamsi adamsi + D. adamsi drumonti 26 0.006 (0–0.017)
D. yui yui 3 0.011 (0.002–0.017) 0.062 (0.056–0.073)
D. dabryi 5 0.008 (0–0.015) 0.150 (0.130–0.162) 0.140 (0.130–0.149)
D. uenoi katoi 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)
D. wallichii bowringi 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)
D. w. wallichii 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)
D. w. bourgoini 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)
Protaetia brevitarsis* 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)

Clade A is composed of D. adamsi adamsi, D. a. drumonti, and D. yui yui with strong bootstrap support (>72%). The two subspecies of D. adamsi did not separate into two distinct subgroups. The genetic divergences between the two subspecies were relatively low (0–1.7%); moreover, D. a. drumonti shared haplotypes with D. a. adamsi from Korea and China. D. yui yui was sister to D. adamsi with distinct inter-specific divergences (5.6%–7.3%).

Clade B is composed of D. dabryi, D. uenoi katoi, and three subspecies of D. wallichii with strong bootstrap supports by ML and BI, but relatively low support (56%–62%) by MP. Among the members of Clade B, D. dabryi and D. uenoi katoi formed a monophyletic group with very strong supporting values in all analyses and with distinct inter-specific divergences (5.6%–8.9%). The intra-specific divergences of these two species (0–1.5% in D. dabryi, 0.2%–2.3% in D. u. katoi) were explicitly lower than their inter-specific values. The three subspecies of D. wallichii were clustered as a monophyletic group and clearly subdivided. D. w. bowringi diverged early from an ancestor, and then D. w. wallichii and D. w. bourgoini underwent subsequent separation with strong bootstrap supports by ML (83%) and BI (99%); however, despite low divergences within each subspecies ranging from 0.3%–0.8%, the genetic divergences between these subspecies were unexpectedly variable ranging from 2.7%–8.1%. Genetic divergences were larger between D. w. bowringi and both D. w. wallichii (4.3%–5.0%) and D. w. bourgoini (4.8%–8.1%), than those between D. w. wallichii and D. w. bourgoini (2.7%–5.7%).

Phylogenetic analyses of 16S rRNA

ML, BI, and MP analyses of 16S rRNA resulted in considerably similar topologies to those of COI (Fig. 3 for BI, ML and MP tree data now shown, see Suppl. material 2 for sequences), but a polytomy was found in D. yui yui and paraphyly in D. w. bowringi with respect to D. w. wallichii.

Figure 3.

Phylogenetic relationships among Dicronocephalus species reconstructed with Bayesian inference using 16S rRNA sequences. Numbers above branches indicate ML bootstrap values and Bayesian posterior probabilities. Numbers below branches are bootstrap, symmetric resampling, and jacknife support from parsimony searches, respectively. Scale bar represents 10% nucleotide mutation rate.

The intra-specific pairwise distances of 16S rRNA were relatively low, ranging from 0–0.4%. The inter-specific divergences ranged from 0.8%–6.3%. The distances between the ingroup and outgroup taxa ranged from 9.7%–11.8% (Table 3). The lowest inter-specific divergence range (0.8%–1.2%) was revealed between D. adamsi and D. yui yui, and this is rather similar to the divergence ranges of the D. wallichii subspecies (0.8%–1.6%).

Pairwise distance of 16S ribosomal RNA within and between Dicronocephalus spp.

No. of samples Within species Between subspecies & species
D. a. adamsi + D. a. drumonti D. yui yui D. dabryi D. uenoi katoi D. w. bowringi D. w. wallichii D. w. bourgoini
D. a. adamsi + D. a. drumonti 22 0.000 (0.000–0.002)
D. yui yui 3 0.001 (0.000–0.002) 0.009 (0.008–0.012)
D. dabryi 5 0.002 (0.000–0.004) 0.057 (0.054–0.060) 0.050 (0.046–0.052)
D. uenoi katoi 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)
D. wallichii bowringi 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)
D. w. wallichii 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)
D. w. bourgoini 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)
Protaetia brevitarsis* 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)

D. adamsi was clustered as a sister to D. yui yui in Clade A with strong bootstrap support (>90%), while the remaining taxa were clustered into Clade B with relatively low supporting values (>76%) in BI and MP. The monophyly of D. adamsi, D. uenoi katoi, D. w. wallichii, and D. w. bourgoini was well supported by bootstrap analyses (>84%). In contrast, in all analyses a polytomy was found in D. yui yui and ML and BI showed paraphyly of D. w. bowringi. We showed that these phenomena were caused by few parsimony-informative nucleotide variations in conserved regions. A comparison of each of those sequences, showed that D. y. yui has different substitutions at 326 nucleotide position. Two samples (7290 and 7291) have “C”, while one sample (7292) has “T”. On the other hand, D. w. bowringi has a substitution occurred in 196 nucleotide position. The 7693 sample has “G”, while the other samples (7692, 7694, and 7695) and two samples (7274 and 7275) of D. wallichii have “A” at this site (Suppl. material 2).

Phylogenetic analyses of COI and 16S rRNA

In the combined data set of COI and 16S rRNA, phylogenetic reconstructions produced topologies congruent with the COI analyses. The nodal supporting values were improved compared with the analyses based on each gene (Fig. 4, see Suppl. material 3 for sequences). Monophyly of the seven taxa including subspecies was strongly supported by bootstrap values >90%, except for low support of 53% and 55% in ML and BI, respectively, for the terminal node of D. w. bowringi. D. w. wallichii was grouped as a sister to D. w. bourgoini based on the results of the COI analyses with a high value in BI (94%) and moderate value in ML (74%), but not in MP (Fig. 4).

Figure 4.

Phylogenetic relationships among Dicronocephalus species reconstructed with Bayesian inference using COI and 16S rRNA sequences. Numbers above branches indicate ML bootstrap values and Bayesian posterior probabilities. Numbers below branches are bootstrap, symmetric resampling, and jacknife support from parsimony searches, respectively. Scale bar represents 10% nucleotide mutation rate.

Re-examination of morphological diagnostic characters

The 19 diagnostic characters used to classify species or subspecies were re-examined in order to determine whether they are suitable for identification (Table 4). Of these characters, mentioned in previous studies, 13 are clearly suitable for species or subspecies identification; however, we recognized six characters that are ambiguous and not applicable (Table 5). For example, Pouillaude (1914) mentioned three diagnostic characters as follows: 1) D. dabryi has a different color of the pronotum and the elytra compared with D. wallichii subspecies (Fig. 1); 2) D. w. wallichii can be separated from the others (D. adamsi, D. w. bowringi, D. w. bourgoini, D. dabryi, and D. beiti) by having no angular projection at the base of the anterior edge of the clypeus (Fig. 5); and 3) D. w. bourgoini can be distinguished from the others by the projected apicosutural angle of the elytra (Fig. 6). However, none of these characters has proven to be suitable for species identification. We observed that the color of the pronotum and the elytra of D. dabryi was the same with grayish powder in freshly collected specimens, but it has faded gradually in old specimens (Fig. 1D). Also the anterior edge of the clypeus of D. w. wallichii (Fig. 5G) was sinuate in the middle, similar to that of D. w. bourgoini (Fig. 5H), and did not match the description by Pouillaude. We therefore consider that these characters might have been mistakenly described and illustrated by Pouillaude (1914). In addition, the projection of the apicosutural angle of the elytra of D. w. bourgoini was not distinct and could not separate this taxon from the other species and subspecies (Fig. 6H). We consider that using another character such as “the posterior margin of the elytra is round or truncated” may more diagnostic than the former character as shown in Fig. 6. Pascoe (1863) used the triangular umbone on the shoulder of the elytra (Fig. 7) to distinguish D. a. adamsi from D. w. bowringi. But, we consider that the presence of a triangular umbone is as an unsuitable character. We found this state also in some specimens of D. adamsi, although the size of the triangular umbone was small and variable in each specimen. Kurosawa (1986) used the widest portion of the pronotum as a distinguishing character state, but this was variable in all specimens of D. w. bourgoini and not distinct enough to be used in species and subspecies identification.

Figure 5.

Anterior edge of clypeus of Dicronocephalus. A D. adamsi adamsi B D. a. drumonti C D. yui yui D D. dabryi E D. uenoi katoi F D. wallichii bowringi G D. w. wallichii H D. w. bourgoini.

Figure 6.

Apicosutural angle of Dicronocephalus. A D. adamsi adamsi B D. a. drumonti C D. yui yui D D. dabryi E D. uenoi katoi F D. wallichii bowringi G D. w. wallichii H D. w. bourgoini.

Figure 7.

Umbone (in the circle) of shoulder of Dicronocephalus. A D. adamsi adamsi B D. a. drumonti C D. yui yui D D. dabryi E D. uenoi katoi F D. wallichii bowringi G D. w. wallichii H D. w. bourgoini.

Diagnostic characters of Dicronocephalus.

Character states Reference
Body 1. Color in male (Fig. 1) 0) grayish brown Kurosawa (1968)
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 Kurosawa (1986)
1) not dark blackish body
3. Pronotal and elytral colors (Fig. 1) 0) pronotum and elytra different Pouillaude (1914)
1) pronotum and elytra similar
4. Dorsal surface 0) pilose with brownish semirecumbent hairs Pouillaude (1914)
Kurosawa (1968)
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 Kurosawa (1968)
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 Kurosawa (1968)
1) weakly prominent
2) absent
7. Shape of anterior edge of clypeus (Fig. 5) 0) simple without angular projection Pouillaude (1914)
1) with an angular projection
8. Circular indentation of clypeus 0) with a strong or weak circular indentation on the edge Pouillaude (1914)
1) without circular indentation on the edge
Pronotum 9. Pronotal bands 0) reaching posterior border Pouillaude (1914)
Young (2012)
1) not reaching posterior border
10. Central carinae 0) carinae defined Pascoe (1866)
1) carinae nearly indistinct
11. Extending of carinae 0) extending beyond the middle Kurosawa (1968)
1) never extending beyond the middle
2) no carina
12. The widest portion 0) widest near the middle Kurosawa (1968)
1) widest in front of the middle
Elytra 13. Surface 0) with two black dots Young (2012)
1) without black dot
14. Shoulder (Fig. 6) 0) with triangular umbone Pascoe (1866)
1) without triangular umbone
15. Apicosutural angle (Fig. 7) 0) rounded Pouillaude (1914)
1) projected
Metasternum 16. Metasternal process 0) obtuse, rather rounded Kurosawa (1968)
Young (2012)
1) rectangular or acute, moderately produced
2) triangularly and sharply produced
Abdomen 17. Abdominal sternites in male 0) covered with yellowish grey powder Pouillaude (1914)
1) normal, not covered with yellowish grey powder
Legs 18. Color of tarsi 0) clear reddish brown (=testaceous) Pascoe (1866)
Pouillaude (1914)
Young (2012)
1) black or very dark brown
19. Length of tarsi 0) anterior tarsi of the male about as long as posterior ones Kurosawa (1968)
1) anterior tarsi distinctly longer than the others

Data matrix for Dicronochephalus species in this study.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
D. adamsi adamsi 0 0 1 1 1 ? 1 0 1 1 2 0 1 1 (rarely 0) 0 1 0 1 1
D. adamsi drumonti 0 0 1 1 1 ? 0 0 1 1 2 0 1 1 (rarely 0) 0 1 0 1 1
D. yui yui 1 0 1 1 0 2 0 0 1 1 1 0 (or 1) 1 1 (rerely 0) ? 1 0 1 1
D. dabryi 0 1 0 (or 1) 1 1 ? 1 1 0 1 2 1 0 1 (rarely 0) 0 0 1 1 1
D. uenoi katoi 1 1 1 0 0 2 1 0 0 1 2 1 1 1 ? 0 1 1 0
D. w. bowringi 3 1 1 1 1 0 (or 1) 0 (or 1) 1 1 0 0 0 1 0 0 1 1 0 1
D. w. wallichii 2 1 1 1 1 0 0 (or 1) 1 1 0 0 0 1 0 0 2 1 0 1
D. w. bourgoini 4 1 1 1 1 1 1 1 1 0 1 1 (rarely 0) 1 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

Legrand (2005) used six diagnostic characters to distinguish between the two subspecies, D. a. adamsi and D. a. drumonti. Among them, we found four characters, namely body size, general body shape, longitudinal bands on the pronotum, and the shape of the triangular umbone of the elytra, to be ambiguous. He also illustrated the metasternal process and the parameres and explained in the key to subspecies that the ridge of the metasternal process does not reach the plate, and the process is weakly raised and more rounded anteriorly in D. a. drumonti. Also, the parameres of D. a. drumonti are shorter and with more acute lateral angles than of D. a. adamsi. However, we found that these characters were variable in the specimens from the two geographically isolated populations (Fig. 8). For example, the shape of the lateral angles of the parameres of Tibetan D. a. drumonti (Fig. 8C, D) is similar to that of a D. a. adamsi from South Korea (Fig. 8K, L), and another specimen of D. a. drumonti from Sichuan, China (Fig. 8G, H) resembles a D. a. adamsi from Dandong, China (Fig. 8S, T). We did not find any significant diagnostic characters to separate the two subspecies and therefore the new synonymy is here proposed (Dicronocephalus adamsi drumonti Legrand, 2005 = Dicronocephalus adamsi adamsi Pascoe, 1863, syn. nov).

Figure 8.

Metasternal process (in the circle) and aedeagi of Dicronocephalus adamsi drumonti and D. a. adamsi. A, B, C, D D. a. drumonti (Tibet) E, F, G, H D. a. drumonti (Sichuan) I, J, K, L D. a. adamsi (South Korea) M, N, O, P D. a. adamsi (North Korea) Q, R, S, T D. a. adamsi (Dandong, China).

Discussion

From the results inferred from ML, BI, and MP methods using COI and 16S rRNA genes, the genus Dicronocephalus includes two major lineages, one with D. adamsi and D. yui yui and another with D. dabryi, D. uenoi katoi, D. w. bowringi, D. w. wallichii, and D. w. bourgoini (Figs 13). The specimens of eight taxa including subspecies clustered into seven groups and their monophyly was strongly supported in all analyses. However, D. w. bowringi was found to be paraphyletic and the monophyly of D. yui yui was not confirmed in the 16S rRNA based analyses. In the same analyses we also failed to identify the monophyly of D. yui yui (Fig. 3). Paraphyly or polytomy of the two species was the result of a few pasimony-informative nucleotide substitutions. This has a significant effect on phylogenetic reconstructions when the genetic divergences within and between species are low.

In all topologies, D. adamsi is sister to D. yui yui; the same was suggested by Kurosawa (1986). He grouped D. adamsi, D. shimomurai, and D. yui as the adamsi species-group and mentioned that the female dark blackish body without markings might be the main characteristic of this group. The abdomen covered with whitish powder is also a trait that is only shared by D. adamsi and D. yui among the examined species (Pouillude 1914, Kurosawa 1986).

In contrast with the molecular data of the adamsi species-group, our results for the other congeners do not support the view of Kurosawa (1986). D. uenoi katoi is treated as a separate group in his paper, but it appears a sister taxon of D. dabryi in our study, although the general appearance of D. uenoi katoi is rather similar to that of D. yui yui. Especially, these two species share two characters: the pronotal bands reaching the posterior border and the obtuse metasternal process. Pouillaude (1914) also noted that D. dabryi has tawny erect hair on the pronotum and elytra. We could observe that the pronotum and elytra are sparsely pilose and the hairs are much denser and longer on the ventral side compared with the other congeners. Furthermore, in the male genitalia, the parameres of the two species are similar and much shorter than those of other species. In this study, the pilose body, which is represented as a unique character of D. uenoi katoi by Kurosawa (1986), is considered as autapomorphy, which may have been rapidly acquired during allopatric speciation in Taiwan because D. uenoi katoi was isolated from a continental ancestor. This interpretation disagrees with Kurosawa’s presumption that D. uenoi katoi is the most primitive in this genus.

Regarding the status of the subspecies of D. adamsi, Legrand (2005) recognized discontinued distribution and morphological differences between two geographically separated populations; however, we consider almost all of the diagnostic characters as being unsuitable for distinguishing these two subspecies. Furthermore, the molecular data indicates that the two subspecies form a monophyletic group with low genetic divergences (0–1.7%) and individuals of the both subspecies share haplotypes. Therefore, our results provide strong evidence that D. a. drumonti should be synonymized with D. a. adamsi.

The three subspecies of D. wallichii were originally described as separate species (Hope 1831, Pascoe 1863, Pouillaude 1914). Subsequently their status was lowered to subspecific (Paulian 1960, Mikšić 1971, 1977, Krajcik 1998, Sakai and Nagai 1998, Šípek et al. 2008, Young 2012, Krajcik 2014). However, Kurosawa (1968) disagreed with Paulian (1960) as he considered that there were significant morphological differences between them such as the characteristics of the antlers, the clypeus, the marginal carinae of the pronotum, and the metasternal process. Devecis (2008) also proposed that the taxa be restored as species based on the morphological differences such as color of the dorsal setation, shape of the antlers, and length of the pronotal bands. Results of our molecular analyses showed that the three subspecies of D. wallichii form a monophyletic group with high supporting values and large genetic distances. The average pairwise distances (4.7%–6.0%) of COI between D. wallichii bowringi + D. wallichii wallichii and D. wallichii bowringi + D. wallichii bourgoini. D. wallichii wallichii + D. wallichii bourgoini were slightly lower than the average inter-specific distances of D. adamsi + D. yui yui (6.2%) and D. dabryi + D. uenoi katoi (6.9%) (Table 2). Also, in 16S rRNA analysis, the pairwise distances between the three subspecies of D. wallichii were similar to (0.8%–1.6%) the distance between D. adamsi and D. yui yui (0.8%–1.2%) (Table 3). Our phylogenetic analyses explicitly explain their evolutionary history. D. w. bowringi is the most primitive among this group and D. w. wallichii might be separated by parapatric speciation in the continental region. Also, D. w. bourgoini might have undergone allopatric speciation after colonizing the volcanic island of Taiwan. Our results support specific rather than subspecific rank of the three members of D. wallichii. We revealed them as being in a monophyletic cluster (Mishler and Theriot 2000, Wiens and Penkrot 2002) with each other separated by distinct genetic gaps in the COI and COI+16S analyses, although not in the 16S rRNA analysis. Also, our study showed two distinguishable morphological characters, namely the color of the dorsal body side in males and the shape of the metasternal process (Table 5). However, this evidence is not strong enough to propose specific rank for each of them. A recent study showed that the high genetic divergence of COI alone cannot be a reason for species separation in Cetonia aurata aurata (Ahrens et al. 2013). There is a need for additional analyses with representative sample sizes and the use of multiple genetic loci to reconfirm our results.

Acknowledgements

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

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