Genes Genet. Syst. (2016) 91, p. 209–215Divergence times of the giant rhinoceros 209 Cretaceous origin of giant rhinoceros beetles (Dynastini; Coleoptera) and correlation of their evolution with the Pangean breakup

Haofei Jin1, Takahiro Yonezawa1,2*, Yang Zhong1, Hirohisa Kishino3 and Masami Hasegawa1,2 1School of Life Sciences, Fudan University, SongHu Rd. 2005, Shanghai 200438, China 2Institute of Statistical Mathematics, Midori-cho 10-3, Tachikawa, Tokyo 190-8562, Japan 3Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

(Received 8 January 2016, accepted 30 April 2016; J-STAGE Advance published date: 23 August 2016)

The giant rhinoceros beetles (Dynastini, , Coleoptera) are distributed in tropical and temperate regions in Asia, America and Africa. Recent molecular phylogenetic studies have revealed that the giant rhinoceros beetles can be divided into three clades representing Asia, America and Africa. Although a correlation between their evolution and the continental drift during the Pangean breakup was suggested, there is no accurate divergence time estimation among the three clades based on molecular data. Moreover, there is a long chronological gap between the timing of the Pangean breakup (Cretaceous: 110–148 Ma) and the emergence of the oldest fossil record (Oligocene: 33 Ma). In this study, we estimated their divergence times based on molecular data, using several combinations of fossil calibration sets, and obtained robust estimates. The inter-continental divergence events among the clades were estimated to have occurred about 99 Ma (Asian clade and others) and 78 Ma (American clade and African clade), both of which are after the Pangean breakup. These estimates suggest their inter-continental divergences occurred by overseas sweepstakes dispersal, rather than by vicariances of the population caused by the Pangean breakup.

Key words: , molecular phylogeny, time tree, fossil calibrations

attempted to reconstruct their phylogenetic tree with INTRODUCTION extensive taxon sampling until recently (Rowland and The tribe Dynastini, known as the giant rhinoceros Miller, 2012). beetles, includes some of the largest species within the Rowland and Miller (2012) inferred the phylogenetic Coleoptera, which is the most species-rich order among all tree on the basis of morphological characters as well living organisms. These species show remarkable sexual as molecular data including mitochondrial and nuclear dimorphism, such as very long horns in the male’s head gene sequences. They indicated that this tribe can and pronotum that are usually absent in females (Endrödi, be divided into three subtribes, Dynastina (Dynastes, 1985). Naturalists have been attracted for centuries by Golofa, Megasoma, Augosoma), Xylotrupina (, such marked morphological variety and excess male orna- , Trypoxylus, Xyloscaptes) and Chalcosomina mentation (Rowland and Miller, 2012). It is important to (Haploscapanes, Chalcosoma, Beckius, Eupatorus, clarify how this sexual ornamentation evolved, to gain a Pachyoryctes) (Supplementary Table S1). The first sub- better understanding of natural selection and especially tribe consists of the African clade (Augosoma) and the sexual selection. To elucidate this issue, reconstruction New World clade (Dynastes, Golofa, Megasoma). On the of a reliable phylogenetic tree is prerequisite. Never- other hand, the latter two subtribes are distributed in theless, no molecular studies of the Dynastini had been Asia and Oceania, and constitute a large clade (here- after called the Asian clade). From this categoriza- Edited by Hidenori Nishihara tion, Rowland and Miller (2012) suggested a correlation * Corresponding author. E-mail: [email protected] Abbreviations: ML (maximum likelihood), Ma (mega annum), between Dynastini evolution and the Pangean breakup in OKR (oldest known record) the Early Cretaceous. DOI: http://doi.org/10.1266/ggs.16-00003 Indeed, the correlations between continental drift and 210 H. JIN et al. phylogenetic branching patterns of terrestrial tide substitutions among genes and codon positions. The such as Eutherian mammals (Nishihara et al., 2009; dos best partition was found by the PartitionFinder program Reis et al., 2012), Palaeognathae (ratite and tinamou: (Lanfear et al., 2012). The best partition is shown in Cooper et al., 2001; Haddrath and Baker, 2012; Yonezawa et Supplementary Table S2, and it was applied for both the al., 2017), Pleurodira (side-necked turtle: Vargas-Ramírez phylogenetic inference and the divergence time estima- et al., 2008), Anura (frogs: Bossuyt et al., 2006), Cichlidae tions under the GTR+Γ model. The phylogenetic tree (cichlid fish: Friedman et al., 2013) and Plecoptera was inferred by the maximum likelihood (ML) method (stoneflies: McCulloch et al., 2016) have been vigorously using the RAxML program ver. 8.0.20 (Stamatakis et al., argued. Some of these studies (e.g., Haddrath and Baker, 2008). The rapid bootstrap method (Stamatakis et al., 2012; dos Reis et al., 2012; Yonezawa et al., 2017) sug- 2008) was applied to evaluate the confidence of the inter- gested that biogeographical distribution patterns were not nal branches with 1000 replications. Divergence times necessarily established merely by continental drift, but were estimated by the Bayesian relaxed clock method that the overseas ‘sweepstakes dispersal route’ (Simpson, (Thorne et al., 1998) with the MCMCTREE program 1940) should be considered. A sweepstakes dispersal implemented in PAML ver. 4.7 (Yang, 2007; Inoue et al., route is “a possible route of faunal interchange which is 2010). To reduce the computational burden, the normal unlikely to be used by most animals, but which will, by approximation method was applied. The autocorrelated chance, be used by some” (Allaby, 1999). Since we focus rates model (Thorne et al., 1998) and the independent on the establishment of intercontinental distribution pat- model (Rannala and Yang, 2007) were used for the drift terns, hereafter we call them ‘overseas sweepstakes dis- model of the evolutionary rates along the branches. The persal’ as an alternative to the hypothesis that vicariance prior distributions of the root rates were set to 4 and 2.26 was caused by the Pangean breakup. for the α and β parameters of Γ distribution, respectively, In addition, no paleontological evidence supports the and the parameters of σ2 were set to 1 and 0.2. Other hypothesis that the breaking up of Pangea triggered the parameters such as the Birth and Death rate were set to speciation of the subfamily Dynastinae because their default. The condition of the MCMC was as follows: the fossil record can be traced back to after the Oligocene first 200,000 generations were discarded as burnin; then, (Krell, 2000, 2007). Therefore, reliable divergence times 20,000 trees were sampled per 200 generations. within the Dynastini based on molecular data are impor- The history of morphological character traits was tant for a better understanding of the correlation between inferred using MESQUITE ver. 3.3 (http://mesquiteproject. their evolution and geological events such as continen- org) with the maximum parsimony method on the basis of tal drift. Here, we present the first clear picture of the the 17 characters reported by Rowland and Miller (2012). time-scale of giant rhinoceros evolution and its cor- relation with geological events by detailed reanalyses of Fossil calibrations Reliable fossil records of the family Rowland and Miller (2012)’s molecular and morphological Scarabaeidae (Krell, 2000, 2007) were used as calibration data. points. The age of the oldest known record (OKR) of a fossil in a particular crown group does not necessarily correspond to the exact timing of its emergence because MATERIALS AND METHODS of the incompleteness of the fossil record. Therefore, the Phylogenetic inference and divergence time estima- emergence timing of the crown group should be assumed tions Detailed procedures for tree inference and diver- between the OKR of crown taxa and the OKR of the stem gence time estimation were as follows. The nucleotide taxa (Yonezawa et al., 2007). Here we suggest the cali- sequence data published by Rowland and Miller (2012) brations of divergence times (maximum and minimum (cytochrome c oxidase II, 16S rRNA, histone III and arginine boundaries of the nodes) in the Dynastini based on this kinase) were retrieved from NCBI. Rowland and Miller idea. The first calibration is the split between the sub- (2012)’s data cover all genera among the tribe Dynastini. families Dynastinae and Melolonthinae (node 2 in Fig. In addition, the corresponding nucleotide sequences of 1). The subfamily Dynastinae (Dynastini species, Tribolium castaneum (family Tenebrionidae) were also and Cyclocephala) is closer to the subfamily Cetoniinae included as an outgroup. The accession numbers are pre- (Cotinus mutabilis) than to the subfamily Melolonthinae sented in Supplementary Table S1. Retrieved sequences (Polyphylla decemlineata). This relationship is sup- were automatically aligned by the MAFFT program (Katoh ported by morphological (Krell, 2000) and molecular data and Standley, 2013), and carefully checked by eye. The (Rowland and Miller, 2012, this study). The reliable impact of the partition model on both tree inference and OKR of Melolonthinae (Cretomelolontha transbaikalica: divergence time estimation was discussed in detail in our Fossil 1 in Fig. 1) is from Baysa, Zazinskaya Forma- previous studies (Omoto et al., 2009; Wu et al., 2014). The tion, Transbaykalia, Russia (Nikolajev, 1998; Krell, 2000, unlinked partition model (Pupko et al., 2002) was applied 2007). This formation belongs to the Valanginian stage to take account of the different tempo and mode of nucleo- (132.9–139.8 Mega annum (Ma; million years ago)) of Divergence times of the giant rhinoceros beetles 211

Fossil 4 199.3-201.3 Ma Tribolium castaneum Tenebrionidae Polyphylla decemlineata Melolonthinaeae Fossil 1 132.9-139.8 Ma Cyclocephala sp. TrypoxylusTrypoxylus dichotodichotomm Fossil 5 29.6 237 -242 Ma 9 XyloscaptesXyloscaptes davidisdavidis 49.849.8 221.2221.2 XXylotrupesylotrupes australicusaustralicus 8 31.9 1 43.543.5 11 XXylotrupesylotrupes meridionalismeridionalis 102.8102.8 10 68.0686 .0 AAllomyrinallomyrina pfeifferpfeifferii 5 7 Chalcosoma caucasus Fossil 3 20.8 152.1-157.3 Ma 13 CChalcosomahalcosoma atlas 54.954.9 140.2140.2 HaHaploscapanesploscapanes barbarossbarbarossaa 12 2 51.751.7 Eupatorus ggracilicornisracilicornis 27.427.4 98.598.5 14 16 6.9669.9 PachyoryctesPachyoryctes sosoliduslidus Dynastinae 6 49.149.1 17 EupatorusEupatorus birmanicubirmanicuss 15 BBeckiuseckius beccabeccariirii 111.3111.3 AAugosomaugosoma centaurucentauruss 4 Golofa pporteriorteri 11.9 77.577.5 33.933.9 21 GolofaGolofa pizarrpizarroo 18 20 GGolofaolofa claviclavigeragera 66.066.0 DDynastesynastes tityustityus 131.2131.2 19 5.45.4 3 52.252.2 23 DDynastesynastes ggrantiranti 22 36.836.8 MeMegasomagasoma acteoacteonn 24 MeMegasomagasoma thersitethersitess

Orizabus clunalis Fossil 2 (about 34 Ma) Cotinus mutabilis Cetoniinae OKR of Cetoniinae (about 17 Ma)

Boreotheria (Laurasia) 83.8 89.1

AAfrotheriafrotheria (A(Afrfriicca)a)

87.1 XenarthraXenarthra (South AmAmerericica)a) 69.4 Pangea Breakup (148 - 110 Ma)

Early Middle Late Early Middle Late Early Late Paleocene Eocene Oligocene Miocene Plio. Pleisto. Holo.

Triassic Jurassic Cretaceous Paleogene Neogene Quaternary

Morphological character state transformation

1 Nodal number

250 200 150 100 50 0 Fossil information 98.5 Divergence time

195Ma 94Ma Modern Fig. 1. The result of divergence time estimation. A time-calibrated phylogenetic tree of the Dynastini as inferred from mitochon- drial genes (COII and 16S) and nuclear genes (H3 and ArgKin). The time-scaled tree and divergence times were estimated with the autocorrelated model. The horizontal axis indicates geological time in Ma. Nodal bars indicate the 95% CIs of the divergence time estimations. Numbers in circles near the nodes are the individual nodal numbers of the Dynastini that are used in Tables 1 and 2, Supplementary Fig. S1 and Supplementary Table S3. The species names of Asian clade, African clade and American clade members are indicated in the pale green box, the pale blue box and the pale orange box, respectively. Nodal bars colored in purple indicate the divergence events among Asian, African and American clades, while those colored in green and in orange indicate the emergence times of the Asian and American clade, respectively. Details of Fossils 1–5 as well as the OKR of the Cetoniinae are described in the main text, and also correspond with Table 1. Gray circles on branches indicate changes in morphological character states (see Supplementary Fig. S1 for details). The outline of the time-calibrated phylogenetic tree of Eutherian mammals (dos Reis et al., 2012) is also shown for comparison, and the colors of the nodal bars are concordant with those of the Dynastini. Fossil 4 (Elaterophanes) belongs to the family Elateridae, and not the family Tenebrionidae; however, since Elateridae are phylogenetically closer to Tenebrionidae than to Scarabaeidae, Fossil 4 represents the Elateridae+Tenebrionidae clade, and is therefore indicated on the branch of Tenebrionidae. The image of Tribolium castaneum (top) is from Wikipedia. The copyright of the Dynastini images belongs to Mushi-sha Ltd., and the images are used with their permission. The photos of the Eutherian mammals (Panthera tigris, Loxodonta africana and Myrmecophaga tridactyla) were taken by M. Hasegawa. the Lower Cretaceous. On the other hand, the OKRs of Krell, 2007; Fossil 3 in Fig. 1) from Karatau, Kazakhstan, Dynastinae and Cetoniinae can be traced back to only after and these fossils are thought to be of Kimmeridgian age the Oligocene (Fossil 2 in Fig. 1). Therefore, the minimum (152.1 to 157.3 Ma) in the Late Jurassic. Therefore, the boundary of the Dynastinae-Melolonthinae split (node 2) tentative maximum boundary of this split (node 2) was should be 132.9 Ma. Generally, the maximum boundary assumed to be 157.3 Ma. The second calibration is the of the divergence time is difficult to set, because the OKRs split of the family Scarabaeidae and the family Tenebri- of the stem taxa do not guarantee the emergence of the onidae (Tribolium castaneum) (node 1 in Fig. 1). Because crown taxa, especially when a long ghost lineage exists the family Elateridae is closer to the family Tenebrionidae or a stem taxon survives for a very long time period as than to the family Scarabaeidae (Hunt et al., 2007), the a relic (Yonezawa et al., 2007; Benton et al., 2009). The OKR of Elateridae (Elaterophanes: Fossil 4 in Fig. 1) from OKR of the whole family Scarabaeidae is Antemnacrassa Dorset, U.K., identified as Hettangian age (199.3–201.3 spp. or Juraclopus rohdendorfi (Nikolajev, 2005; see also Ma) in the Early Jurassic (Whalley, 1985; Hunt et al., 212 H. JIN et al.

2007), can be used as the minimum boundary. On the (character 17 in Rowland and Miller, 2012) evolved most other hand, the OKR (Fossil 5 in Fig. 1) of the Coleoptera frequently (5 steps). Except for this character, male orna- (Cupedidae) is known from Madygen, Fergana, Kyrgyzstan mentation (2.44 steps on average) evolved significantly and identified as Ladinian age (237–242 Ma) in the Middle faster than other characters (1.43 steps on average) (t-test: Triassic (Arnol’di et al., 1992; Hunt et al., 2007). This P = 0.037). It is not surprising that sexual selection age can be tentatively used as the maximum boundary of should be the strongest driving force of the morphological this node. These calibrations are summarized in Table 1. variety in this tribe. At the same time, the reconstructed ancestral states suggest that convergent evolution fre- Evaluation of the prior distributions by Bayes fac- quently occurs in sexual ornamentation. tor Although the prior distributions of the divergence There was no significant correlation between the mor- times (e.g., drift model for changing of the evolutionary phological evolutionary rates and the molecular evolution- rates along the branches, fossil calibrations) are important, ary rates along the branches (data not shown). normally these are evaluated by circumstantial evidence, especially consistency and inconsistency with the fossil Divergence times The result of the divergence time esti- record (e.g., Zhong et al., 2009; Wu et al., 2015). Lepage mation is shown in Fig. 1 and Table 2. The autocorrelated et al. (2007) suggested the application of the Bayes factor model was selected for the drift model of the evolutionary to evaluate drift models (autocorrelated model and inde- rate (log Bayes factor (BF) ≈ 40: strong support), but selec- pendent model). Here, we expand this idea to evaluate tion of these two models gave a very limited effect on the not only the drift models, but also fossil calibrations by estimates (Table 2). The choice of fossil calibrations did the Bayes factor with Tracer ver. 1.6 (http://tree.bio.ed.ac. not have any considerable effect on the estimates. Exclu- uk/software/tracer/). sion of the calibration on the Scarabaeidae-Tenebrionidae split (node 1 in Fig. 1) made the marginal likelihood score higher (ln BF = 1.05 – 1.39), but ln BF values of < 3 are RESULTS AND DISCUSSION considered negligible (Kass and Raftery, 1995). It sug- Phylogenetic tree The ML tree of the Dynastini is gests that the assumption of the maximum boundary on shown in Supplementary Fig. S1. The branching patterns their split is not appropriate, and that the actual diver- are essentially the same as those of Rowland and Miller gence time of this node (Scarabaeidae-Tenebrionidae split) (2012)’s tree except for the basal position of Chalcosoma is older than this maximum boundary. within the subtribe Chalcosomina in this study, and the To test the hypothesis that the subfamily Dynastinae nodal support values are generally higher. The evolu- emerged in the Oligocene, we also estimated the divergence tionary history of 17 morphological characters described times under each of the following two assumptions. The by Rowland and Miller (2012) was traced on the basis first assumption asw that the divergence between the of this ML tree topology (Supplementary Figs. S1 and subfamily Dynastinae and its sister subfamily Cetoniinae S2). Among the 17 characters, dorsal integument color occurred during the Oligocene, and the second assumption

Table 1. Fossil calibrations

Nodal ln Marginal Maximum time2 (Ma) Minimum time2 (Ma) number1 likelihood3

Calibration 1 242 [fossil 5: Cupedidae] 199.3 [fossil 4: Elaterophanes] −134.65 set 1 2 157.3 [fossil 3: Antemnacrassa and Juraclopus] 133 [fossil 1: Cretomelolontha transbaikalica] Calibration 2 157.3 [fossil 3: Antemnacrassa and Juraclopus] 133 [fossil 1: Cretomelolontha transbaikalica] −133.26 set 2 Calibration 1 242 [fossil 5: Cupedidae] 199.3 [fossil 4: Elaterophanes] −133.61 set 3 1 242 [fossil 5: Cupedidae] 199.3 [fossil 4: Elaterophanes] Calibration 2 157.3 [fossil 3: Antemnacrassa and Juraclopus] 133 [fossil 1: Cretomelolontha transbaikalica] −139.46 set 4 3 33.9 [fossil 2: Ligyrus, , Strategus] 23.3 [fossil 2: Ligyrus, Pentodon, Strategus] 1 242 [fossil 5: Cupedidae] 199.3 [fossil 4: Elaterophanes] Calibration 2 157.3 [fossil 3: Antemnacrassa and Juraclopus] 133 [fossil 1: Cretomelolontha transbaikalica] −138.52 set 5 4 33.9 [fossil 2: Ligyrus, Pentodon, Strategus] 23.3 [fossil 2: Ligyrus, Pentodon, Strategus] 1 Nodal numbers correspond to those of Fig. 1 and Supplementary Fig. S1. 2 Numbers of the fossils are concordant with Fig. 1. 3 ln Marginal likelihood scores were estimated by the harmonic mean. Divergence times of the giant rhinoceros beetles 213

Table 2. Estimated divergence times

Autocorrelated model Independent model Node1 Posterior mean Ma 95% CI Node1 Posterior mean Ma 95% CI 1 221.2 200.9–231.1 1 219.7 198.8–231.0 2 140.2 132.5–153.6 2 140.6 132.5–154.0 3 131.2 111.9–148.5 3 129.2 106.2–148.4 4 111.3 86.7–135.6 4 109.7 82.4–136.2 5 102.8 80.1–125.6 5 99.1 74.3–125.5 6 98.5 76.6–120.2 6 94.1 70.5–119.6 7 68.0 49.1–88.6 7 60.2 42.3–81.4 8 49.8 34.4–68.2 8 43.3 29.5–60.6 9 29.6 17.9–44.7 9 24.9 13.6–39.8 10 43.5 29.2–61.0 10 37.1 24.4–53.1 11 31.9 19.6–47.6 11 27.1 15.1–42.4 12 5 4.9 37.9–74.1 12 47.1 30.6–66.9 13 20.8 11.1–33.5 13 16.8 7.9–28.7 14 51.7 35.3–70.4 14 43.9 27.8–63.2 15 49.1 32.9–67.7 15 41.4 25.7–60.4 16 27.4 12.9–46.3 16 21.4 8.0–41.3 17 6.9 2.0–13.9 17 4.9 1.0–10.6 18 77.5 56.9–99.8 18 79.8 56.6–105.9 19 66.0 46.6–87.6 19 68.7 47.3–94.0 20 33.9 20.3–51.2 20 40.0 22.9–61.8 21 11.9 5.7–20.4 21 15.1 6.4–26.6 22 52.2 34.6–72.9 22 53.7 34.5–77.2 23 5.4 1.6–10.8 23 5.6 1.3–11.4 24 36.8 21.8–55.6 24 37.9 20.2–60.3 ln Marginal likelihood2 = −134.65 ln Marginal likelihood2 = −174.13 1 Nodal numbers correspond to Fig. 1 and Supplementary Fig. S1. 2 ln Marginal likelihood scores were estimated by the harmonic mean. was that the crown group of the subfamily Dynastinae Dynastinae species other than Dynastini are insufficiently emerged during the Oligocene (the split of Orizabus and accumulated in public databases, the effect of taxon sam- other members of the Dynastini was assumed to have pling in the outgroup should be examined in a future occurred in the Oligocene (23.0 Ma to 33.9 Ma)). These study. In every case of prior distributions in terms of assumptions made the marginal likelihood scores lower rate drift models as well as fossil calibrations (except (ln BF = 3.87 – 4.80), indicating that the exclusion of such for the ‘bad’ assumption that the subfamily Dynastinae assumptions results in positive effects (20 > ln BF > 3: emerged in the Oligocene), the estimated divergence Kass and Raftery, 1995). These results suggest that our time (node 6 in Fig. 1, 98.5 Ma, 95% CI 76.6–120.2 Ma) evaluation of the fossil calibration is objective. between Dynastina (African-New World clade) and The effect of taxon sampling on divergence time esti- Xylotrupina+Chalcosomina (Asian clade), which is also mation was also investigated. Sparser taxon sampling the time of the most recent common ancestor (tMRCA) of typically had no considerable effect (except for the esti- the tribe Dynastini, falls within the late Early Cretaceous mate of Dynastes and Megasoma in data set 2) (Supple- to the early Late Cretaceous (94.1–114.0 Ma). The diver- mentary Table S3). However, in one case, when the gence time between the African clade and the New World taxon samplings were dense in the outgroup and sparser clade (node 18 in Fig. 1) was estimated to be 56.9–99.8 Ma in the ingroup, the estimates became grossly younger (the Late Cretaceous). Even considering the instability (please refer to Supplementary Table S3 and Supplemen- caused by taxon sampling, these dates are within the late tary Table S4 for details of the members of the ingroup Early to the Late Cretaceous, and the Late Cretaceous to and outgroup). Since the nucleotide sequence data of the Paleocene, respectively. Since the breakup of Pangea 214 H. JIN et al. is thought to have occurred around 110 to 148 Ma (Early changes in the time-scaled tree (Fig. 1) suggests that Cretaceous), and more specifically 120 Ma (Nishihara et the rates accelerated episodically in the Paleocene to al., 2009), the initial diversification of the tribe Dynastini Eocene. These accelerated rates in morphological evo- seems to have occurred during or just after the Pangean lution are harmonious with the episodic adaptive radia- breakup. tion after the mass extinction. Explosive emergence of The tMRCA of the New World clade (node 19) is 46.6– higher taxonomic groups after the mass extinction, at both 87.6 Ma, and that of the Asian clade (node 7) is 49.1–88.6 molecular and morphological levels, is known in several Ma. This means that the tribe Dynastini had already clades such as the Eutherian mammals (molecular level: diversified in the Late Cretaceous to the Eocene. Since dos Reis et al., 2012; morphological level: O’Leary et al., fossils of the subfamily Dynastinae first emerged in the 2013) and the Aves (molecular level: Jarvis et al., 2014; Oligocene (Krell, 2000, 2007), as mentioned above, there morphological level: Clark et al., 2005). These findings are very long ghost lineages. It is notable that the phy- will contribute to a better understanding of faunal suc- logeographical branching pattern and divergence times cessions after mass extinctions. are compatible with those of the Eutherian mammals Besides the tribe Dynastini, the subfamily Dynastinae (Fig. 1). Overseas sweepstakes dispersal just after the includes the seven tribes Agaocephalini, Oryctini, Pangean breakup can be considered to be a major driv- Phileurini, , Oryctoderini, Cyclocephalini and ing force for the establishment of a distribution pat- Hexodontini. Some tribes (e.g., Oryctini, Pentodontini) tern. However, considering the large standard errors are globally distributed, and some (e.g., Oryctoderini, in our estimates for the separation between Asian and Hexodontini) are regionally distributed. Phylogenetic African+New World lineages (node 6, 76.6–120.2 Ma) and relationships among these tribes are poorly understood that between African and New World (node 18, 56.9–99.8 (but see the morphological analysis by Endrödi, 1985). In Ma) lineages, the breaking up of Pangea itself also should addition, how their sexual dimorphism and ornamentation be taken into consideration. evolved, and how they established their distribution area, According to Allaby (1999), sweepstakes disper- are interesting issues. Molecular phylogenetic analyses sal “requires a major barrier that is occasionally based on more extensive taxon sampling of this subfamily crossed. Which groups cross and when they cross are will shed more light on these issues in the future. determined virtually at random.” Since the distances among Africa, South America and Eurasia in the Late We wish to express our special thanks to Dr. Jun Adachi for his Cretaceous were shorter than they now are (Nishihara support. This study was supported by a Grant-in-Aid for Scien- et al., 2009), it is plausible that distribution areas were tific Research (No. 25440219) from JSPS to M. H., and by NSFC 30925004 to Y. Z. We also thank Mushi-Sha Ltd. (Nakano, established by overseas sweepstakes dispersal across the Tokyo, Japan) for providing the images of the Dynastini. We short intercontinental distances during the Cretaceous. deeply acknowledge the helpful comments by two anonymous As several species of the Asian clade (, reviewers and the handling editor. Allomyrina pfeifferi, or the genus Haploscapanes) are distributed in Australasia beyond the Wallace Line, if REFERENCES the distances between the landmasses were sufficiently short, these species could expand their distribution Allaby, M. (1999) A Dictionary of Zoology. Oxford University Press, Oxford, UK. areas. However, at the same time, if such overseas dis- Arnol’di, L. V., Zherikhin, V. V., Nikritin, L. M., and Ponomarenko, persal occurred frequently, the phylogeographic pattern A. G. (1992) Mesozoic Coleoptera. Nauka Publishers, would be disturbed. Considering the very clear relation- Moscow, Russia. ships between the phylogenetic branching patterns and Benton, M. J., Donoghue, P. C. J., and Asher, R. J. (2009) Calibrat- geographic distribution patterns, such overseas dispersal ing and constraining molecular clocks. In: The Timetree of Life (eds.: Hedges, S. B., and Kumar, S.), pp. 35–86. Oxford events are likely to have been very rare. University Press, New York, USA. Our results suggest that all major Dynastini lineages Bossuyt, F., Brown, R. M., Hillis, D. M., Cannatella, D. C., and emerged in the Paleogene, especially during the Paleocene Milinkovitch, M. C. (2006) Phylogeny and biogeography of a and Eocene (Fig. 1), despite their poor fossil record. The cosmopolitan frog radiation: Late Cretaceous diversification Paleocene and Eocene can be characterized as the warm resulted in continent-scale endemism in the family Ranidae. Syst. Biol. 55, 579–594. period in the Cenozoic era (Zachos et al., 2001). Because Clarke, J. A., Tambussi, C. P., Noriega, J. I., Erickson, G. M., most of the Dynastini species are adapted to tropical and Ketcham, R. A. (2005) Definitive fossil evidence for the regions, it is plausible that they adapted to various empty extant avian radiation in the Cretaceous. Nature 433, ecological niches after the K-Pg extinction, and diversi- 305–308. fied at the morphological level thereafter. Interestingly, Cooper, A., Lalueza-Fox, C., Anderson, S., Rambaut, A., Austin, J., and Ward, R. (2001) Complete mitochondrial genome although morphological evolutionary rates did not show sequences of two extinct moas clarify ratite evolution. significant correlation with estimated time intervals Nature 409, 704–707. (Supplementary Fig. S3), the distribution pattern of the dos Reis, M., Inoue, J., Hasegawa, M., Asher, R. J., Donoghue, Divergence times of the giant rhinoceros beetles 215

P. C., and Yang, Z. (2012) Phylogenomic datasets provide ous divergence of the three superorders of mammals. Proc. both precision and accuracy in estimating the timescale of Natl. Acad. Sci. USA 106, 5235–5240. placental mammal phylogeny. Proc. Biol. Sci. London 279, O’Leary, M. A., Bloch, J. I., Flynn, J. J., Gaudin, T. J., Giallombardo, 3491– 3500. A., Giannini, N. P., Goldberg, S. L., Kraatz, B. P., Luo, Z. Endrödi, S. (1985) The Dynastinae of the World. W. Junk Pub- X., Meng, J., et al. (2013) The placental mammal ancestor lishers, The Hague, The Netherlands. and the post-K-Pg radiation of placentals. Science 339, Friedman, M., Keck, B. P., Dornburg, A., Eytan, R. I., Martin, C. 662–667. H., Hulsey, C. D., Wainwright, P. C., and Near, T. J. (2013) Omoto, K., Yonezawa, T., and Shinkawa, T. (2009) Molecular sys- Molecular and fossil evidence place the origin of cichlid tematics and evolution of the recently discovered “Parnas- fishes long after Gondwanan rifting. Proc. R. Soc. B 280, sian” butterfly (Parnassius davydovi Churkin, 2006) and its 20131733. http://dx.doi.org/10.1098/rspb.2013.1733. allied species (Lepidoptera, Papilionidae). Gene 441, 80–88. Haddrath, O., and Baker, A. J. (2012) Multiple nuclear genes Pupko, T., Huchon, D., Cao, Y., Okada, N., and Hasegawa, M. and retroposons support vicariance and dispersal of the (2002) Combining multiple data sets in a likelihood analysis: palaeognaths, and an Early Cretaceous origin of modern which models are the best? Mol. Biol. Evol. 19, 2294–2307. birds. Proc. Biol. Sci. London 279, 4617–4625. Rannala, B., and Yang, Z. (2007) Inferring speciation times under Hunt, T., Bergsten, J., Levkanicova, Z., Papadopoulou, A., John, an episodic molecular clock. Syst. Biol. 56, 453–466. O. S., Wild, R., Hammond, P. M., Ahrens, D., Balke, M., Rowland, J. M., and Miller, K. B. (2012) Phylogeny and systemat- Caterino, M. S., et al. (2007) A comprehensive phylogeny ics of the giant rhinoceros beetles (Scarabaeidae: Dynastini). of beetles reveals the evolutionary origins of a superradia- Insecta Mundi 0263, 1–15. tion. Science 318, 1913–1916. Simpson, G. G. (1940) Mammals and land bridges. J. Wash. Inoue, J., Donoghue, P. C., and Yang, Z. (2010) The impact of the Acad. Sci. 30, 137–163. representation of fossil calibrations on Bayesian estimation Stamatakis, A., Hoover, P., and Rougemont, J. A. (2008) A rapid of species divergence times. Syst. Biol. 59, 74–89. bootstrap algorithm for the RAxML Web servers. Syst. Biol. Jarvis, E. D., Mirarab, S., Aberer, A. J., Li, B., Houde, P., Li, C., 57, 758–771. Ho, S. Y., Faircloth, B. C., Nabholz, B., Howard, J. T., et al. Thorne, J. L., Kishino, H., and Painter, I. S. (1998) Estimating (2014) Whole-genome analyses resolve early branches in the the rate of evolution of the rate of molecular evolution. Mol. tree of life of modern birds. Science 346, 1320–1331. Biol. Evol. 15, 1647–1657. Kass, R. E., and Raftery, A. E. (1995) Bayes factors. J. Am. Stat. Vargas-Ramíreza, M., Castaño-Morab, O. V., and Fritz, U. (2008) Assoc. 90, 773–795. Molecular phylogeny and divergence times of ancient South Katoh, K., and Standley, D. M. (2013) MAFFT multiple sequence American and Malagasy river turtles (Testudines: Pleurodira: alignment software version 7: improvements in performance Podocnemididae). Org. Divers. Evol. 8, 388–398. and usability. Mol. Biol. Evol. 30, 772–780. Whalley, P. E. S. (1985) The systematics and palaeogeography Krell, F. T. (2000) The fossil record of Mesozoic and Tertiary of the Lower Jurassic of Dorset, England. Bull. Br. Scarabaeoidea (Coleoptera: Polyphaga). Invertebr. Taxo. Mus. Nat. Hist. (Geol.) 39, 107–189. 14, 871–905. Wu, J., Hasegawa, M., Zhong, Y., and Yonezawa, T. (2014) Impor- Krell, F. T. (2007) Catalogue of fossil Scarabaeoidea (Coleoptera: tance of synonymous substitutions under dense taxon Polyphaga) of the Mesozoic and Tertiary, Version 2007. sampling and appropriate modeling in reconstructing the Denver Museum of Nature & Science Technical Report mitogenomic tree of Eutheria. Genes Genet. Syst. 89, 2007-8, 1–79. 237–251. Lanfear, R., Calcott, B., Ho, S. Y., and Guindon, S. (2012) Parti- Wu, J., Kohno, N., Mano, S., Fukumoto, Y., Tanabe, H., tionfinder: combined selection of partitioning schemes and Hasegawa, M., and Yonezawa, T. (2015) Phylogeographic substitution models for phylogenetic analyses. Mol. Biol. and demographic analysis of the Asian black bear (Ursus Evol. 29, 1695–1701. thibetanus) based on mitochondrial DNA. PLoS ONE 10, Lepage, T., Bryant, D., Philippe, H., and Lartillot, N. (2007) A e0136398. doi:10.1371/journal.pone.0136398. general comparison of relaxed molecular clock models. Mol. Yang, Z. (2007) PAML 4: phylogenetic analysis by maximum like- Biol. Evol. 24, 2669–2680. lihood. Mol. Biol. Evol. 24, 1586–1591. McCulloch, G. A., Wallis, G. P., and Waters, J. M. (2016) A time- Yonezawa, T., Nikaido, M., Kohno, N., Fukumoto, Y., Okada, N., calibrated phylogeny of southern hemisphere stoneflies: and Hasegawa, M. (2007) Molecular phylogenetic study on Testing for Gondwanan origins. Mol. Phylogenet. Evol. 96, the origin and evolution of Mustelidae. Gene 396, 1–12. 150–160. Yonezawa, T., Segawa, T., Mori, H., Campos, P. F., Hongoh, Y., Nikolajev, G. V. (1998) Vidy plastinchatousykh zhukov gruppy Endo, H., Akiyoshi, A., Kohno, N., Nishida, S., Wu, J., et al. Pleurosticti (Coleoptera, Scarabaeidae) iz nizhnego mela (2017) Phylogenomics and morphology of extinct paleog- Zabaikaliya. Paleontologicheskii Zhurnal 1998, 77–84. naths reveal the origin and evolution of the ratites. Curr. Nikolajev, G. V. (2005) Novyy rod podsemeystva Aclopinae Biol. 27, 68–77. (Coleoptera, Scarabaeidae) iz verkhney yury Kazakhstana. Zachos, J., Pagani, M., Sloan, L., Thomas, E., and Billups, K. Trudy Instituta Zoologii, Ministerstvo Obrazovaniia i Nauki (2001) Trends, rhythms, and aberrations in global climate Respubliki, Zoologoekologicheskie Issledovaniya 49, 112– 65 Ma to present. Science 292, 686–693. 114. Zhong, B., Yonezawa, T., Zhong, Y., and Hasegawa, M. (2009) Epi- Nishihara, H., Maruyama, S., and Okada, N. (2009) Retroposon sodic evolution and adaptation of chloroplast genomes in analysis and recent geological data suggest near-simultane- ancestral grasses. PLoS One 4, e5297.