Research Article Journal of Orthoptera Research 2017, 26(1): 11-19 Phylogenetic relationship of Japanese Podismini species (Orthoptera: Acrididae: Melanoplinae) inferred from a partial sequence of cytochrome c oxidase subunit | gene BEATA GRrZYWACz!, HARUKI TATSUTA! 1 Department of Ecology and Environmental Sciences, Faculty of Agriculture, University of the Ryukyus, Nishihara, Okinawa, Japan. 2 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Stawkowska 17, 31-016 Krakow, Poland. Corresponding author: B. Grzywacz (grzywacz@isez.pan.krakow.pl) Academic editor: Corinna Bazelet | Received 19 July 2016 | Accepted 4 July 2017 | Published 28 June 2017 http://zoobank.org/B899 16BC-A54B-4B92-97DE-9D 1 BFO3BE8A6 Citation: Grzywacz B, Tatsuta H (2017) Phylogenetic relationship of Japanese Podismini species (Orthoptera: Acrididae: Melanoplinae) inferred from a partial sequence of cytochrome c oxidase subunit I gene. Journal of Orthoptera Research 26(1): 11-19. https://doi.org/10.3897/jor.26.14547 Abstract Members of the tribe Podismini (Orthoptera: Acrididae: Melanopli- nae) are distributed mainly in Eurasia and the western and eastern regions of North America. The primary aim of this study is to explore the phy- logenetic relationship of Japanese Podismini grasshoppers by comparing partial sequences of cytochrome c oxidase subunit I (COI) mitochondrial gene. Forty podismine species (including nineteen Japanese species) and thirty-seven species from other tribes of the Melanoplinae (Dactylotini, Dichroplini, Melanoplini, and Jivarini) were used in the analyses. All the Japanese Podismini, except Anapodisma, were placed in a well-supported subclade. However, our results did not correspond with the classification on the basis of morphological similarity for the status of Tonkinacridina. This group of Japanese species constituted a single clade with other spe- cies of Miramellina and Podismina, while Eurasian continental species of Tonkinacridina were placed in other separate clades. This incongruence might have resulted from historical migratory events between continent and ancient islands and subsequent convergent/parallel evolution in mor- phology. Some remarks on phylogenetic positions in Podismini and other tribes were also made in terms of reconstructed phylogeny. Key words grasshoppers, polymorphism, mitochondrial DNA Introduction The tribe Podismini Jacobson, 1905 is one of the five tribes belonging to the acridid subfamily Melanoplinae Scudder, 1897 (Cigliano et al. 2017). Podismini genera are distributed in the Palearctic and Nearctic region (Vickery 1987). They usually occur in grassland and scrub formations. Although morphology is rather variable between species, most species are clearly definable (Ito 2015). According to morphological features, Podismini is currently divided into three subtribes: Miramellina (Rehn & Randell, 1963), Podismina (Jacobson, 1905), and Tonkinacridina (Ito, 2015). The genus group Bradynotae (Rehn & Randell, 1963) and another 21 genera have also been considered as members of this tribe, but they have not yet been included in the subtribes (Cigliano et al. 2017). Because of the substantial variability in morphology and even in karyotype, the taxonomy of Podismini has been excessively confused. Based on the reexamination of characters, Japanese Podismini consists of 22 species in nine genera (Ito 2015), while the phylogenetic relationship between species in the tribe is still ambiguous. The first molecular study of Podismini examined one mitochondrial gene (COI) and three ribosomal nuclear and mitochondrial genes (ITS1, 12S, and 16S) for 25 species of Podismini (Chintauan-Marquier et al. 2014). In this study, nine Japanese species of seven genera [Parapodisma dairisama (Scudder, 1897), P. mikado (Bolivar, 1890), P. subastris Huang, 1983; Sinopodisma punctata Mistshenko, 1954; Ognevia longipennis (Shiraki, 1910); Podisma kanoi (Storozhenko, 1994); Zubovskya koeppeni parvula (Ikonnikov, 1911); Fruhstorferiola okinawaensis (Shiraki, 1930); Tonkinacris sp. (Carl, 1916)] were also examined. Four of seven genera (Parapodisma Mistshenko, 1947, Sinopodisma Chang, 1940, Tonkinacris Carl, 1916, Fruhstorferiola Willemse, 1921) constituted a clade with moderate statistical support, two of seven (Podisma Berthold, 1827, Ognevia Ikonnikov, 1911) composed another clade, and Zubovskya Dovnar-Zapolsky, 1932 did not comprise a clade with any other genera. The Japanese archipelago is composed ofa multitude of smaller islands in addition to the four main islands (Hokkaido, Honshu, Kyushu, and Shikoku). The isolation of the Japanese archipelago from the Eurasian continent presumably began in Miocene (ca. 23 Myr ago), and the present form of the archipelago was reached in the approximate end of Pleistocene (lijima and Tada 1990, Tada 1994, Yonekura et al. 2001). Interestingly, land bridges between the continent and some of the islands were formed at least three times during geochronologic periods between the Pliocene and Pleistocene as a result of changes in sea level during ice ages (Dobson and Kawamura 1998), which may have permitted back- and-force movement of animals via the bridges. These complex geological events have probably shaped the present fauna and flora in Japan. The present Japanese Podismini had also presumably been derived in part from continental species group which evolved uniquely at a new place. JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1) 12 Sinopodisma The Japanese archipelago in broad sense consists of Hokkaido, Honshu, Shikoku, Kyushu, south Kuril Islands, and chain of islands extending from southwestern Kyushu to northern Taiwan (i.e. “Nansei Islands”). The brief distribution of nine genera in Podismini is shown in Fig. 1. Three of nine genera, Fruhstorferiola, Sinopodisma, and Tonkinacris are distributed only in Nansei Islands, presumably derived directly from continental species of the same genera. The genus Anapodisma Dovnar-Zapolskii, 1933 is found only in Tsushima Island, the southern vicinities of Korean Peninsula. The distribution of Prumna Motschulsky, 1859, Zubovskya, and Podisma is localized in a northern part of Japan (central - northern Honshu, Hokkaido and Kunashir Island) and the habitat tends to be highly fragmented especially in mountain districts. Ognevia shows the broadest distribution range among Japanese Podismini and is distributed in high altitude areas. Although other genera are apterous or have reduced forewings, flight organs are fully developed in this genus. The genus Parapodisma comprises 11 species (50% of the Japanese podismine species) including two subspecies in Japan, and shows a variety of morphology such as body colors, genitalic characters and forewings, which has sometimes confounded their taxonomic status (Ito 2015). Although Vickery (1977) suggested that Sinopodisma, Fruhstorferiola and Parapodisma comprise Miramellina together with Zubovskya and Miramella Dovnar-Zapolskii, 1933, B. GRZYWACZ AND H. TATSUTA w-zzs, Lubovskya af” “-<— Fruhstorferiola ob ,” ~ : ew é ; ge Tonkinacris La Figure 1. A map of Japan with the distribution of nine genera of Japanese Podismini. Ito (2015) proposed that the first three genera with Tonkinacris should be settled in a new subtribe, Tonkinacridina based on the cladistic assessment of 23 morphological traits. The principal aim of the present study is to examine whether Ito’s (2015) hypothesis still holds if the relationship is assessed using mitochondrial DNA sequences. We utilized a partial sequence of the cytochrome c oxidase subunit I (COI) mitochondrial gene for this purpose because the sequence is used as standard in DNA barcoding and thus is feasible for comparing species other than Japanese Podismini. In order to test the hypothesis of a close affinity between all Japanese taxa, other Melanoplinae species from Eurasia and America were also drawn from GenBank and included in the analysis. Materials and methods Taxa studied.— A total of 82 species and subspecies were included in the analysis. All genetic sequences were acquired from GenBank except Podismini species in Japan (Table 1). The in-group consis- ted of 20 Podismini species and subspecies from Japan (new data) and 21 species from Eurasia and America. We included members of four other tribes of Melanoplinae: Melanoplini (19 species), Dactylotini (3 species), Dichroplini (13 species), and Jivarini (2 species). As an outgroup, we included four species of subfam- JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1) B. GRZYWACZ AND H. TATSUTA Table 1. Taxonomic information and GenBank accession numbers for taxa included in this study. Taxa outgroup Subfamily: Catantopinae Xenocatantops humilis (Serville, 1838) Catantops erubescens (Walker, 1870) Diabolocatantops innotabilis (Walker, 1870) Goniaea vocans (Fabricius, 1775) Subfamily: Melanoplinae Tribe: Dactylotini Dactylotum bicolor bicolor Charpentier, 1845 Liladownsia fraile Fontana, Marino-Pérez, Woller & Song, 2014 Perixerus squamipennis Gerstaecker, 1873 Tribe: Dichroplini Atrachelacris unicolor Giglio-Tos, 1894 Atrachelacris gramineus Bruner, 1911 Baeacris pseudopunctulata (Ronderos, 1964) Chlorus bolivianus Brunner, 1913 Dichromatos lilloanus (Liebermann, 1948) Dichroplus obscurus Bruner, 1900 Dichroplus pratensis Brunner, 1900 Leiotettix pulcher Rehn, 1913 Neopedies noroestensis Ronderos, 1991 Pseudoscopas nigrigena (Rehn, 1913) Ronderosia bergii (Stal, 1878) Ronderosia forcipata (Rehn, 1918) Scotussa daguerrei Liebermann, 1947 Tribe: Jivarini Jivarus americanus Giglio-Tos, 1898 Jivarus gurneyi Ronderos, 1979 Tribe: Melanoplini Hypochlora alba (Dodge, 1876) Melanoplus bivittatus (Say, 1825) Melanoplus borealis (Fieber, 1853) Melanoplus bowditchi Scudder, 1878 Melanoplus bruneri Scudder, 1897 Melanoplus cinereus Scudder, 1878 Melanoplus dawsoni (Scudder, 1875) Melanoplus deceptus Morse, 1904 Melanoplus differentialis (Thomas, 1865) Melanoplus femurrubrum (De Geer, 1773) Melanoplus gladstoni Scudder, 1897 Melanoplus infantilis Scudder, 1878 Melanoplus mexicanus (Saussure, 1861) Melanoplus montanus (Thomas, 1873) Melanoplus oregonensis (Thomas, 1875) Melanoplus packardii Scudder, 1878 Melanoplus punctulatus (Uhler, 1862) Melanoplus sanguinipes (Fabricius, 1798) Phoetaliotes nebrascensis (Thomas, 1872) Sampling locality and year China Pakistan Pakistan Australia North America North America North America South America South America South America, Argentina South America South America South America South America, Argentina South America, Argentina South America South America South America, Argentina South America, Argentina South America, Argentina South America South America North America, USA North America, Canada North America, Canada North America, Canada North America, Canada North America, Canada North America, Canada North America, Canada North America North America, Canada North America, Canada North America, Canada North America North America, Canada North America, Canada North America, Canada North America, Canada North America, Canada North America, Canada JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1) Accession No. EU366111 KJ672128 KJ672135 JX033911 KJ531421 KJ531423 KJ531427 FJ829334 AY014360 DQ083452 FJ829333 FJ829336 DQ084357 DQ083459 DQ083464 AF539852 FJ829342 DQ083467 DQ083468 DQ083469 DQ389233 DQ389231 AF260548 KR141481 KR142429 KM535226 KM535553 KR141925 KM537453 KR140464 KJ531425 KM536630 KR140625 KM537809 KJ531426 KM536558 KR140837 KM537414 KR140511 KR143225 KM535800 13 Reference Wang and Jiang (unpublished) Nazir et al. (unpublished) Nazir et al. (unpublished) Chapco 2013 Woller et al. 2014 Woller et al. 2014 Woller et al. 2014 Dinghi et al. 2009 Amédégnato et al. 2003 Colombo et al. 2005 Dinghi et al. 2009 Dinghi et al. 2009 Dinghi et al. 2009 Colombo et al. 2005 Colombo et al. 2005 Ameédégnato et al. 2003 Dinghi et al. 2009 Colombo et al. 2005 Colombo et al. 2005 Colombo et al. 2005 Chapco 2006 Chapco 2006 Chapco et al. 2001 Hebert et al. 2016 Hebert et al. 2016 Dewaard et al. (unpublished) Dewaard et al. (unpublished) Hebert et al. 2016 Dewaard et al. (unpublished) Hebert et al. 2016 Woller et al. 2014 Dewaard et al. (unpublished) Hebert et al. 2016 Dewaard et al. (unpublished) Woller et al. 2014 Dewaard et al. (unpublished) Hebert et al. 2016 Dewaard et al. (unpublished) Hebert et al. 2016 Hebert et al. 2016 Dewaard et al. (unpublished) 14 Taxa Tribe: Podismini Subtribe: Miramellina Anapodisma beybienkoi Rentz & Miller, 1971 Anapodisma miramae Dovnar-Zapolskij, 1932 Zubovuskya koeppeni parvula (Ikonnikov, 1911) Zubovuskya koeppeni parvula (Ikonnikov, 1911) Zubovuskya koeppeni parvula (Ikonnikov, 1911) Zubovuskya koeppeni parvula (Ikonnikov, 1911) Subtribe: Podismina Ognevia longipennis (Shiraki, 1910) Ognevia sergii Ikonnikov, 1911 Podisma kanoi Storozhenko, 1994 Podisma kanoi Storozhenko, 1994 Podisma sapporensis Shiraki, 1910 Podisma sapporensis Shiraki, 1910 Podisma tyatiensis Bugrov & Sergeev, 1997 Yunnanacris yunnaneus (Ramme, 1939) Subtribe: Tonkinacridina Fruhstorferiola huayinensis Bi & Xia, 1980 Fruhstorferiola kulinga (Chang, 1940) Fruhstorferiola okinawaensis (Shiraki, 1930) Fruhstorferiola okinawaensis (Shiraki, 1930) Fruhstorferiola tonkinensis (Willemse, 1921) Parapodisma awagatakensis Ishikawa, 1998 Parapodisma awagatakensis Ishikawa, 1998 Parapodisma caelestis Tominaga & Ishikawa, 2001 Parapodisma caelestis Tominaga & Ishikawa, 2001 Parapodisma caelestis Tominaga & Ishikawa, 2001 Parapodisma dairisama (Scudder, 1897) Parapodisma dairisama (Scudder, 1897) Parapodisma dairisama (Scudder, 1897) Parapodisma dairisama (Scudder, 1897) Parapodisma mikado (Bolivar, 1890) Parapodisma niihamensis hiurai Tominaga & Kano, 1987 Parapodisma niihamensis niihamensis Inoue, 1979 Parapodisma niihamensis niihamensis Inoue, 1979 Parapodisma niihamensis niihamensis Inoue, 1979 Parapodisma setouchiensis 1 Inoue, 1979 Parapodisma setouchiensis 1 Inoue, 1979 Parapodisma setouchiensis 2 Inoue, 1979 Parapodisma setouchiensis 2 Inoue, 1979 Parapodisma setouchiensis 2 Inoue, 1979 Parapodisma setouchiensis 3 Inoue, 1979 Parapodisma subastris 1 Huang, 1983 Parapodisma subastris 1 Huang, 1983 B. GRZYWACZ AND H. TATSUTA Sampling locality and year Tsushima, Nagasaki, Japan, 2016 China Mt. Daisetsu, Hokkaido, Japan, 2015 Mt. Daisetsu, Hokkaido, Japan, 2015 Mt. Daisetsu, Hokkaido, Japan, 2015 Mt. Daisetsu, Hokkaido, Japan, 2015 China Russia Mt. Yokote, Nagano, Japan, 2014 Mt. Yokote, Nagano, Japan, 2014 Kamishihoro, Hokkaido, Japan, 2015 Nukabira, Hokkaido, Japan, 2015 Russia China China China Kunigami, Okinawa, Japan, 1998 Kunigami, Okinawa, Japan, 1998 China Kanaya, Shizuoka, Japan, 2015 Kanaya, Shizuoka, Japan, 2015 Mt. Kamikouchi, Nagano, Japan, 2016 Mt. Kamikouchi, Nagano, Japan, 2016 Mt. Kamikouchi, Nagano, Japan, 2016 Kofu, Tottori, Japan, 2005 Kofu, Tottori, Japan, 2005 Kofu, Tottori, Japan, 2005 Kofu, Tottori, Japan, 2005 Kami-sugo, Furukawa, Japan Kawachi-nagano, Osaka, Japan, 2015 Yoshinogawa, Tokushima, Japan, 2015 Yoshinogawa, Tokushima, Japan, 2015 Yoshinogawa, Tokushima, Japan, 2015 Mima, Tokushima, Japan, 2015 Mima, Tokushima, Japan, 2015 Minamiasakawa, Hachioji, Japan, 2015 Sefuriyama, Fukuoka, Japan, 2015 Sefuriyama, Fukuoka, Japan, 2015 Toyooka, Hyogo, Japan, 2014 Oe, Kyoto, Japan, 2014 Oe, Kyoto, Japan, 2014 Accession No. KY558890 KM362650 KX440513 KX440514 KX440515 KX440516 JQ301452 KC261364 KX440484 KX440485 KY558881 KY558882 KC261368 KX223964 KC139873 KC139885 KX440482 KY558871 KC139890 KY558873 KY558874 KY558875 KY558876 KY558877 KX440478 KX440479 KX440480 KX440481 KY558878 KX440483 KX440486 KX440487 KX440488 KX440498 KX440499 KX440489 KX440490 KX440491 KY558872 KX440494 KX440495 Reference This study Kang et al. 2016 This study This study This study This study Lit and Huang 2012 Bugrov et al. (unpublished) This study This study This study This study Bugrov et al. (unpublished) Guan and Xu (unpublished) Huang et al. 2013 Huang et al. 2013 This study This study Huang et al. 2013 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1) B. GRZYWACZ AND H. TATSUTA Taxa Sampling locality and year Parapodisma subastris 2 Huang, 1983 Parapodisma subastris 2 Huang, 1983 Parapodisma subastris 2 Huang, 1983 Parapodisma subastris 2 Huang, 1983 Parapodisma tenryuensis 1 Kobayashi, 1983 Parapodisma tenryuensis 1 Kobayashi, 1983 Parapodisma tenryuensis 2 Kobayashi, 1983 Parapodisma tenryuensis 2 Kobayashi, 1983 Parapodisma tenryuensis 2 Kobayashi, 1983 Parapodisma yasumatsui Yamasaki, 1980 Parapodisma yasumatsui Yamasaki, 1980 Sinopodisma aurata Ito, 1999 Sinopodisma aurata Ito, 1999 Sinopodisma houshana Huang, 1982 Sinopodisma kodamae (Shiraki, 1910) Sinopodisma kodamae (Shiraki, 1910) Sinopodisma lofaoshana (Tinkham, 1936) Sinopodisma lushiensis Zhang, 1994 Sinopodisma punctata Mistshenko, 1954 Sinopodisma punctata Mistshenko, 1954 Sinopodisma punctata Mistshenko, 1954 Sinopodisma punctata Mistshenko, 1954 Sinopodisma punctata Mistshenko, 1954 Sinopodisma punctata Mistshenko, 1954 Sinopodisma rostellocerna You, 1980 Sinopodisma tsinlingensis Zheng, 1974 Sinopodisma wulingshanensis Bi, Huang & Liu, 1992 Tonkinacris ruficerus Ito, 1999 Tonkinacris ruficerus Ito, 1999 Tonkinacris yaeyamaensis Ito, 1999 genus group Bradynotae Asemoplus montanus (Bruner, 1885) Bradynotes obesa (Thomas, 1872) 15 a Reference Oe, Kyoto, Japan, 2014 KX440496 This study Oe, Kyoto, Japan, 2014 KX440497 This study Oe, Kyoto, Japan, 2014 KX440492 This study Oe, Kyoto, Japan, 2014 KX440493 This study Oyama, Shizuoka, Japan, 2015 KY558883 This study Oyama, Shizuoka, Japan, 2015 KY558884 This study Mt. Chausu, Shizuoka, Japan, 2016 KY558885 This study Mt. Chausu, Shizuoka, Japan, 2016 KY558886 This study Mt. Chausu, Shizuoka, Japan, 2016 KY558887 This study Sefuriyama, Fukuoka, Japan, 2015 =KX440500 This study Mitsuse, Saga, Japan, 2015 KX440501 This study Kohama Island, Okinawa, : Japan, 2016 KY558888 This study Kohama Island, Okinawa, mapa, 2016 KY558889 This study China KE139919 Huang et al. 2013 Kukuan, Taiwan, 1998 KX440502 This study Kukuan, Taiwan, 1998 KX440503 This study China KC139936 Huang et al. 2013 China KC139925 Huang et al. 2013 Tatsugo, Kagoshima, Japan, 1997 KX440504 This study Tatsugo, Kagoshima, Japan, 1997 KX440505 This study Tatsugo, Kagoshima, Japan, 1997 KX440506 This study Tatsugo, Kagoshima, Japan, 1997 KX440507 This study Tatsugo, Kagoshima, Japan, 1997 KX440508 This study Tatsugo, Kagoshima, Japan, 1997 KX440509 This study China KC139947 Huang et al. 2013 China KC139903 Huang et al. 2013 China KC139909 Huang et al. 2013 Kunigami, Okinawa, Japan, 1998 KX440510 This study Kunigami, Okinawa, Japan, 1998 KX440511 This study Mt. Omoto, Okinawa, Japan, 1998 KX440512 This study North America, Canada Other members of Podismini - do not assign into any subtribe Prumna arctica (Zhang & Jin, 1985) Prumna fauriei (Bolivar, 1890) Prumna fauriei (Bolivar, 1890) Prumna mandshurica Ramme, 1939 Prumna primnoa (Motschulsky, 1846) Qinlingacris choui Li, Wu & Feng, 1991 KM535587 Dewaard et al. (unpublished) North America KJ531419 Woller et al. 2014 China KE139971 Huang et al. 2013 Mt. Gassan, Yamagata, Japan, 2014. + KY558879 This study Mt. Gassan, Yamagata, Japan, 2014. + KY558880 This study China FJ531676 Zhao etal. (unpublished) Russia KX272717 Sukhikh et al. (unpublished) China FJ531684 Zhao etal. (unpublished) ily Catantopinae [Xenocatantops humilis (Serville, 1838), Catantops erubescens (Walker, 1870), Diabolocatantops innotabilis (Walker, 1870), and Goniaea vocans (Fabricius, 1775)]. We did not include Japanese species of the genus Ognevia; instead, an existing se- quence for O. longipennis from China was examined in this paper (Li and Huang 2012). DNA extraction, amplification, and sequencing.— Total genomic DNA was extracted with the DNeasy Tissue Kit (QIAGEN, Hilden, Germany). Partial gene sequences were amplified by PCR using the following primers: forward UEA7 (TACAGTTGGAATAGACGTTGATAC) and reverse UEA10 (TCCAATGCACTAATCIGCCATATTA) (Lunt et al. 1996). PCR was conducted in a 20 pl volume containing 1 pl of DNA, 2 pl 10 x Ex Taq Buffer (Mg** free; Takara Bio Inc., Shiga, Japan) with 10 1M each primer, 10 mM dNTPs, 25 mM MgCl, and 5 U/l of Ex Taq polymerase (Takara Bio Inc., Shiga, Japan). The mitochondrial COI fragment was amplified under the following temperature profile: initial activation at 94 °C for 3 min, 30 cycles of denaturation at JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1) 16 94 °C for 1 min, annealing at 45 °C for 1 min, and elongation at 72 °C for 2 min, and a final elongation step at 72 °C for 7 min. PCR products were purified by using the NucleoSpin Extract II kit (Macherey-Nagel, Diiren, Germany). Samples were sequenced in both directions by using the same primers as those used for PCR and the chain termination reaction method (Sanger et al. 1977). The sequencing was carried out in a total volume of 10 pl by using the Genome Lab Dye Terminator Cycle Sequencing with Quick Start Kit (Beckman Coulter, Brea, California, USA), with a cycle-sequencing profile of 40 cycles of 96 °C for 20 s, 50 °C for 20 s, and 60 °C for 3 min. Sequencing was performed using GenomeLab GexXP™ (Beckman Coulter, Brea, California, USA) at the Laboratory of Entomology in the Faculty of Agriculture, University of the Ryukyus, Japan. Sequences were deposited in GenBank under the accession numbers provided in Table 1. Sequence alignment and phylogenetic analyses.— DNA sequences were aligned by using MUSCLE (Edgar 2004) with default pa- rameters. In order to identify numts (Bensasson et al. 2001, Song et al. 2008), mitochondrial COI sequences were translated into amino acid sequences with MEGA 6 (Tamura et al. 2013) using the standard invertebrate mitochondrial genetic code. The substi- tution model of evolution was estimated by using the program jModelTest (Guindon and Gascuel 2003, Darriba et al. 2013). The Akaike information criterion was preferred over the hierarchical likelihood ratio test to compare the various models as recom- mended by Posada and Buckley (2004). The data matrices were subjected to Bayesian analysis (BI) with MrBayes v3.1. (Huelsen- beck and Ronquist 2001, Huelsenbeck et al. 2001). Bayesian analy- ses were performed with 10 000 000 generations, with a sampling of trees every 100 generations. Likelihood values were observed with Tracer v.1.4 (Rambaut and Drummond 2007); all the trees created before stability in likelihood values were discarded as a ‘burn-in’ (first 1200 trees). Maximum likelihood (ML) analysis was implemented in Phyml (Guindon and Gascuel 2003). For the bootstrapping analyses 1000 pseudoreplicates were generated. FigTree 1.4.0 (Rambaut and Drummond 2012) was used to visual- ize the trees. Results The total alignment of the COI gene consisted of 646 bp in- cluding 53% variable sites and 48% parsimony-informative sites. The analysis of the partial mitochondrial COI gene amplified from 59 individuals revealed 20 different haplotypes. Among them in- dividuals were identical for 14 species except Parapodisma subastris, P. setouchiensis, and P. tenryuensis. The model F81 + G (gamma distribution shape parameter G = 0.6220) was determined to be the most justified. The Bayesian inference and maximum likelihood analyses re- sulted in similar trees, the only differences between them being the degree of statistical support for the recovered nodes (Fig. 2). Nodal supports were generally poor across all backbone nodes. ML bootstrap percentages were lower than BI posterior probabili- ties. The relationship between Podismini and the related tribes were not fully resolved and varied depending on the nodes. Melanoplinae were divided into six distinctive lineages and appeared as a polytomy of four clades (II - VI). Dactylotini (1) was placed as sister to the other five lineages. The second and third lin- eages (II and III) consisted of two genera (seven species) and one genus (three species) of Podismini, respectively. The fourth clade (IV) clustered the genera of Dichroplini. Within clade five (V), B. GRZYWACZ AND H. TATSUTA Melanoplini formed a monophyletic group with strong support [posterior probability (PP) = 1.00, bootstrap value (BV) = 77]. The sixth clade (VI) was constituted of the rest of the members of Po- dismini and Jivarini. Thirteen genera of Podismini included in this study formed three separate clades. The Japanese Podismini, except Anapodisma beybienkoi Rentz & Miller, 1971, were placed in a well supported subclade with high nodal support (PP = 1.00, BV = 100) within clade VI. Nine species of Sinopodisma and four species of Fruhstor- feriola included in the analysis nested in different clades. The ma- jority of Podismini species formed clade VI together with Jivarini species. The basal relationships within clade VI were not resolved. Clade VI consisted of 13 branches with a single terminal taxon: nine species of Parapodisma, Bradynotes obesa (Thomas, 1827), Podisma tyatiensis Bugrov & Sergeev, 1997, Qinlingacris choui Li & Feng, 1991 and Asemoplus montanus (Bruner, 1885), and five sub- clades including members of three podismine subtribes. Tonki- nacridina comprising Parapodisma, Tonkinacris, Fruhstorferiola and Sinopodisma did not constitute a single clade. Among 11 species of Parapodisma, P. tenryuensis Kobayashi, 1983 (two haplotypes), P. caelestis Tominaga & Ishikawa, 2001, P. mikado, and P. awagataken- sis Ishikawa, 1998 were clustered together with moderate statisti- cal support (Fig. 2). Discussion The present study obtained some interesting results with re- spect to the relationships within Japanese Podismini. The subclade of Japanese Podismini within clade VI (indicated with light green frames in Fig. 2) included genera which have been attributed to three podismine subtribes, but Tonkinacridina did not form a sin- gle clade. Two different methodological inferences on phylogeny (BI, ML) yielded mostly congruent nodes, but the trees were poorly resolved (Fig. 2). Most taxa were determined within a large poly- tomy of Podismini, in which only a few clades have been recove- red. Support remained generally low for the deeper nodes, as was expected for a phylogeny constructed using COI only, but some more derived nodes had higher values (Fig. 2). Our results are compared with tree inferred by Chintauan- Marquier et al. (2014) who were the first to show molecular phy- logeny of Eurasian Podismini including nine Japanese species. The most important finding is that Podismini did not constitute monophyly as previously suggested in Chintauan-Marquier et al. (2014), but there are some incongruent patterns between the two. In the present results, most of the species of Japanese Podismini, except Anapodisma, constituted a single clade (Fig. 2), whereas species belonging to Podismina and Miramellina constituted separate clades from Tonkinacridina in the previous molecular study (Chintauan-Marquier et al. 2014). Although the statistical support was not very strong, a monophyly of Tonkinacridina was supported in the previous study, a view concordant with morpho- logical inspection (Ito 2015). On the contrary, our data placed the continental species of Tonkinacridina in different clades (clade II and III in Fig. 2) from Japanese Tonkinacridina. Of course, strict comparisons between these studies are impossible at this stage since continental Tonkinacridina was not included in the previ- ous dataset (Chintauan-Marquier et al. 2014). The view of mono- phyly in Tonkinacridina is quite doubtful. We can postulate that the observed continental and Japanese species of Tonkinacridina assigned in different clusters reflect somewhat historical migration events coupled with geological processes described above and sub- sequent convergent/parallel evolution has eventually accumulated JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1) B. GRZYWACZ AND H. TATSUTA 2.0 17 Jivarus gurneyi JIVARINI Sen Giavie-pet ae Pee L) Prumna mandshurica Prumna arctica Prumna primnoa Asemoplus montanius Qinlingacris choui _ Ognevia longipennis Pedi bets ; SAAT AS BeaMaeneg Tonkinacris ruficerus Tonkinacris yaeyamaensis Prumna fauriei , Zubovskya koeppeni parvula Podisma kanoi P & Podisma sapporensis Sinopodisma punctata == Sinopodisma aurata * Sinopodisma kodamae Bey ley mikado | = Parapodisma tenryuensis 2 PODISMINI Parapodisma caelestis : * Parapodisma awagatakensis Parapodisma tenryuensis 1 Parapodisma subastris1 = = Parapodisma niihamensis hiurai ’ Parapodisma niihamensis nihamensis = Parapodisma yasumatsui — Parapodisma setouchiensis 3 Parapodisma dairisama_ Parapodisma setouchiensis 1 “Parapodisma setouchiensis 2 Parapo BSS subastis, See Beeeeee Dbiad ) anopius Qladsto oplus deceptus ‘Melanoplus punctulatus elanoplus differentialis Phoetaliot galt consi ‘aliotes nebrascensis = Melanoplus borealis Melanoplus dawsoni ~ Melanopius femurrubrum lelanoplus sanguinipes MELANOPLINI et is bolivianus | trachelacnis gramineus Atrachelacris unicolor Ronderosia bergii DICHROPLINI Baeacris pseudopunctulata stl ol bee lilloanus ussa pavers cher PODISMINI DACTYLOTINI outgroup jocatan 2 tantops erubescens _. Diabolocatantops innotabilis Goniaea vocans Figure 2. Phylogenetic tree of Podismini based on the Bayesian analysis (BI) of concatenated COI sequences. BI posterior probability (PP) and maximum likelihood bootstrap values (BV) are shown near resolved branches (only support values above 50% are shown) as PP/BV. The respective clades are marked with a square and Roman numeral. We examined Ognevia longipennis from China because of the availability and thus did not treat this specimen as Japanese Podismini (see also text). Light green frames denote the Japanese Podismini analyzed in the present study. in morphology. This conjecture could be evaluated by estimating coalescent time of clades using a mitochondrial clock. In the genera compared, Parapodisma is particularly interesting because this includes a vast variety of morphological variation in genital and external characters (Kawakami 1999, Kawakami and Tatsuta 2010), while almost no variation in karyotype exists in contrast to morphology (Inoue 1985). Even in the same species, various forms in forewings and body colors are often found and thus have caused synonymous species/subspecies (Kawakami 1999). This taxonomic disorder still continues in this group partly because there is no robust phylogenetic tree that enables to disentangle “genuine” relationships from homoplasy in morphology. Unfortunately, most species constituted polytomy because of a lack of statistical power, a subclade comprised of closely related species, Parapodisma mikado, P. tenryuensis, P. caelestis, and P. awagatakensis was detected (Fig. 2). While P. mikado shows an extended distribution from vicinities of northern Japan and Russia such as Sakhalin, Kunashir, and Hokkaido to the middle of Honshu, the other three species are distributed in narrower regions in Honshu. In particular, populations of P. caelestis are limited to narrow habitats such as flower fields with a variety of wild grass and alpine flora on the top of mountains and P. awagatakensis inhabits patchy forest edges with very low population density; thus are considered to be vulnerable to unexpected environmental degradation. According to the cladistic assessment in morphology, P. awagatakensis was clustered together with P. mikado and P. dairisama, whereas P. tenryuensis constituted holophyly with P. caelestis and P. takeii (Takei, 1914) (this species is not included in our study) (Ito 2015), a result dissimilar to the present molecular relationship. Rigorous character sampling JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1) 18 with additional molecular data is definitely required for resolving the complex relationship between morphological and genetic similarity. We also have to pay attention to possible hybridization between partly sympatric species, while no clear evidence for this has been obtained even in closely related species (Kawakami and Tatsuta 2010). The genus Sinopodisma emerged as a highly paraphyletic group in which species did not appear closely related and nested in dif- ferent clades. Likewise, although Sinopodisma punctata resembles S. kodamae (Shiraki, 1910) in several morphological features such as body color and genital appendages in comparison with S. au- rata Ito, 1999, the inferred tree supports the closer relationship between S. aurata and S. kodamae. Furthermore, most of the con- tinental species of Sinopodisma are distinguished from S. punctata and S. aurata in respect of the features in pronotum and cerci (Ito 2015). We postulate that the morphological similarities within Si- nopodisma are the result of convergent evolution; further intensive studies based on molecular data are definitely necessary for the reliable underpinning of phylogenetic relationships. The present investigation generated additional evidence for the relationships within Melanoplinae. In present trees, Dichroplini spe- cies were recovered as a monophyletic group, in agreement with the analysis of Chapco (2006) and Woller et al. (2014). On the other hand, Chintauan-Marquier et al. (2011) found the paraphyly of Di- chroplini. In our analysis, Dactylotini and Melanoplini species each formed a monophyletic clade. Previous studies of Dactylotini includ- ing Hesperotettix viridis Thomas, 1872 discovered that this tribe is par- aphyletic (Chapco 2006, Chintauan-Marquier et al. 2011, Woller et al. 2014). The prior analysis of the melanopline tribes placed Jivarini in a basal position in the subfamily (Amédégnato et al. 2003, Woller et al. 2014). In our results, Jivarini species were clustered together with Podismini representatives. Different studies (Litzenberger and Chapco 2001, Chintauan-Marquier et al. 2014, Woller et al. 2014) recovered Podismini as a monophyletic group, while Litzenberger and Chapco (2003) hypothesized a paraphyly of Podismini. Although a single mitochondrial gene may lead to a half answer for the whole picture of relationships of higher taxa, the present study provides some significant implications of phylogenetic position. One of the great merits of this study is that the gene has extensively been used for DNA barcoding studies in insects, including grasshoppers, which enables us to examine a store of sequences in a global database (Cameron 2014). The selected mitochondrial COI gene allowed us to estimate intra- and inter- species relationships because of the presence of both variable and conserved regions as well as a heterogeneous evolutionary rate across the gene (Lunt et al. 1996). Simultaneously, we also should keep in mind that the shorter COI gene sequences may include paralogous nuclear mitochondrial pseudogenes (numts) that are apt to induce incorrect inference for phylogenetic relationships (Song et al. 2008, 2014). We need further investigations with orthologous genes for elucidating the distinct phylogenetic position of taxa of interest. Acknowledgments We are greatly indebted to Yasushi Kawakami, Keiichiro Shika- ta, Yoshikazu C. Sugano, Shin-ichi Akimoto, Koji Mizota, Masaka- zu Sano, Yasushi Sato, Takeshi Sasaki, Chiharu Koshio, and Shin- ichi Kudo for their help in collecting materials. Thanks are also due to Gen Ito for enthusiastic discussions and providing valuable materials and documents. Collecting materials at special protec- tion zone in Daisetsuzan National Park and in Southern Alps Na- B. GRZYWACZ AND H. TATSUTA tional Park were approved by the Ministry of Environment of Japan (Nos. 1508181, 1605174, 1605023, 1607014). This work has been supported in part by JSPS KAKENHI Grant Numbers 15F15762, 25291088, and 25304014 to Haruki Tatsuta and Beata Grzywacz. References Amédégnato C, Chapco W, Litzenberger G (2003) Out of South America? Additional evidence for a southern origin of melanoplinae grasshop- pers. 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