Ground Beetles (Coleoptera, Carabidae) of the World Inferred from a Phylogenetic Tree of Mitochondrial ND5 Gene Sequences

Genes Genet. Syst. (2003) 78, p. 53–70 Mode of morphological differentiation in the Latitarsi- ground beetles (Coleoptera, Carabidae) of the world inferred from a phylogenetic tree of mitochondrial ND5 gene sequences

Zhi-Hui Su1*, Yûki Imura2, Hong-Zhang Zhou3, Munehiro Okamoto4 and Syozo Osawa1,† 1JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan 2Shinohara-cho 1249-8, Kohoku-ku, Yokohama 222-0026, Japan 3Institute of Zoology, Chinese Academy of Sciences, Haidian, Beijing 100080, China 4Department of Laboratory Animal Science, School of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan

(Received 19 August 2002, accepted 22 January 2003)

The Latitarsi is one large division of the subtribe Carabina (subfamily Carabi- nae, family Carabidae), and has been considered as a discrete morphological group consisting of 17 genera. The phylogenetic relationships and evolutionary pattern of the Latitarsi ground beetles have been investigated by analyzing mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences. The phylogenetic tree suggests that the Latitarsi members do not form a single cluster, i.e., not mono- phyletic and at least 16 lineages belonging to the so-called Latitarsi emerged at about the same time of the Carabina radiation together with the members of other divisions. This suggests that these lineages (A, B, C, H, L, N, O, P, Q, R, S, T, U, V, W and X in Fig. 2a) may be treated each as a phylogenetically distinct division equivalent to other divisions. The group with bootstrap value of more than 80 percent has been considered as a single lineage (division) with two exceptions, V and X. The independency of each lineage has been assumed by the traditional morphology as well as a single clustering on the trees constructed by independent methods, unchanged topology by replacement of outgroups, etc. Generally speak- ing, the members in a single lineage are geographically linked. Many phyloge- netic lineages are composed of a single or only a few species without conspicuous morphological differentiation. In contrast to such a “silent morphological evolu- tion”, a remarkable morphological differentiation occasionally took place in several lineages.

Key words: ground beetles, Latitarsi, mitochondrial ND5 gene, phylogeny, evo- lution, taxonomy

include 168 species (Brˇezina, l999). The phylogenetic INTRODUCTION trees of the mitochondrial NADH dehydrogenase subunit The Latitarsi are one large taxonomic division of the 5 (ND5) gene sequences have been reported for some ground beetles in the subtribe Carabina, and are widely groups in this division, namely, the Oreocarabus complex distributed throughout the holarctic region and the north- (Imura et al., 1998a), Leptocarabus (Kim et al., 2000a, b) ern periphery of North Africa. Morphologically, they and Tomocarabus in the Japanese Islands (Su et al., have been classified into 17 genera (Imura, l996) which 2000). The representative genera in this division seem to have radiated within a relatively short time a little Edited by Naruya Saiou after or around the radiation of the Carabina 50–40 mil- * Corresponding author. E-mail: [email protected] lion years ago (MYA) as deduced from an ND5 phyloge- † Present address: 2-4-7-1003, Ushita-Asahi, Higashi-ku, netic tree. This division is most probably polyphyletic Hiroshima 732-0067, Japan. (Imura et al., 1998a; Su et al., 2001). This situation The nucleotide sequences data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide seems to make it difficult to estimate the meaningful phy- sequences databases with the accession numbers shown in logenetic positions of various Latitarsi groups. Table 1. In the present study, the NJ-, the UPGMA and the MP-

54 Z.-H. SU et al. phylogenetic trees of the ND5 gene have been constructed MATERIALS AND METHODS for 165 specimens consisting of 77 species of the Latitarsi including 38 species used in the previous papers (Imura The specimens analysed for the ND5 gene sequences in et al., 1998a, b; Su et al., 2001). The overall phylogeny this study are listed in Table l, and the localities where and evolutionary pattern of the Latitarsi ground beetles the samples were collected are shown in Fig. 1. The sci- have been discussed. entific names used by Imura & Mizusawa (1996) and

Table 1. List of the specimens used in this study Specimen DDBJ/EMBL/GenBank No.* Scientific name Locality Accession no. Reference A- 1 Autocarabus auratus auratus France: Bourgogne D86204 Su et al. 1996b 2 Au. cancellatus graniger Romania: Judet Caras-Severin, Teregova env. AB092699 This paper 3 Au. cancellatus graniger Romania: Judet Caras-Severin, Baile Herculane, Topenia Val. AB092700 ” 4 Au. cancellatus tuberculatus Russia: Leningrad Distr., Liebiazhie AB092701 ” 5 Au. cancellatus emarginatus Italy: Piemonte, Vercelli, Crevacuore, near Guardabosone (600 m) AB092702 ” 6 Au. cancellatus carinatus France: Jura AB092703 ” 7 Au. cancellatus carinatus France: Jura AB092704 ” B- 1 Mesocarabus problematicus planiusculus France: Bourgogne D86210 Su et al. 1996b 2 Me. problematicus harcyniae Germany: Niedersachsen, Lamspringe, Hildesheim AB050680 Su et al. 2001 C- 1 Orinocarabus putzeysianus germanae Italy: Piemonte, Cuneo, V. Tanaro, Viozene, Mt. Saccarello (2200 AB017453 Imura et al. m) 1998a 2 Or. putzeysianus omensis Italy: Piemonte, Cuneo, Castelmagno, Col del Mulo (2300 m) AB017454 ” D- 1 Megodontus kolbei aino Japan: Hokkaido, Kamikawa D50365 Su et al. 1996a 2 Aristocarabus viridifossulatus rizeanus China: Sichuan, Jiuzhaigou Xian, Jiuzhaigou, Rizegou (3100 m) AB050677 Su et al. 2001 3 Shenocoptolabrus osawai micangshanus China: Sichuan, Nanjiang, Micang Shan Mts., Daba (1350 m) AB050681 ” E- 1 Gnathocarabus kuznetzovi kuznetzovi Iran: Mazandaran, Gombad-e Kavus, Karim Ishan AB047253 Imura et al. 2000a 2 Archicarabus monticola monticola Italy: Piemonte, Cúneo, Brondello AB047259 ” 3 Ar. nemoralis nemoralis USA: Washington Srat., Snohomish, W. Index AB047262 ” F- 1 Cyclocarabus karaterekensis hemicallis- Kirgiz: SE. of Tshandalash Mts., Tshakmak-Suu Riv. (3100–3400 AB053499 Su et al. 2002 thenes m) 2 Morphocarabus sibiricus haeres Russia: Belgorod, near Borisovka steppe AB053520 ” 3 Mo. venustus liaoningensis China: Liaoning, Dandong, Mt. Shifang Ding AB050678 Su et al. 2001 G- 1 Isiocarabus miaorum miaorum China: Guangxi, Xingían, Mt. Miaoíer Shan AB041053 Tominaga et al. 2000 2 Carabus (s. str.) vanvolxemi vanvolxemi Japan: Aomori, Mutsu, Mt. Osore-zan AB041062 ” H- 1 Cavazzutiocarabus latreilleanus latreil- Italy: V. díAosta, Champorcher (2400 m) AB017478 Imura et al. leanus 1998a I- 1 Limnocarabus clathratus clathratus Germany: W. Niedersachsen, Dalums Moor near Lingen AB031507 Kim et al. 1999a 2 Li. maacki aquatilis Japan: Aomori, Nakasato D50358 Su et al. 1996a 3 Euleptocarabus porrecticollis porrecticol- Japan: Gifu, Nakatsugawa AB022564 Kim et al. lis 1999b J- 1 Hemicarabus tuberculosus tuberculosus Japan: Fukushima, Shirasaka D50353 Su et al. 1996a 2 Homoeocarabus maeander paludis Japan: Hokkaido, Sarobetsu D50354 Su et al. 1996a K- 1 Apotomopterus sauteri yunkaicus China: Guangxi, Mt. Miao’er Shan AB031490 Kim et al. 1999a 2 Ap. tonkinensis tonkinensis Vietnam: Lao Cai Prov., Deo Tram Ton AB031494 ” 3 Ap. iris iris China: Sichuan, Daliang Shan Mts., Meigu~Leibo AB031504 ” L- 1 Autocarabus cristoforii cristoforii France: Vicdesos, Ariège prov. AB092705 This paper M- 1 Chaetocarabus intricatus intricatus France: Bourgogne D86208 Su et al. 1996b 2 Heterocarabus marietti ornatus Turkey: W. Giresun AB047196 Imura et al. 2000b 3 Platycarabus depressus depressus Italy: Piemonte, Varallo, Val Sesia AB047198 ” N- 1 Eurycarabus famini fezzanus Morocco: Michlifen (1800 m) AB092706 This paper

Phylogeny of Latitarsi ground beetles 55

continued O- 1 Nesaeocarabus abbreviatus abbreviatus Spain: Is. Tenerife, El Bueno, Arico (900 m) AB092707 ” 2 Ne. abbreviatus abbreviatus Spain: Is. Tenerife, Siete Fuentes (1500 m) AB092708 ” P- 1 Cytilocarabus cribratus cribratus Turkey: Yusufeli, Artvin AB017460 Imura et al. 1998a 2 Cy. cribratus cribratus Turkey: Coruh, Karcal Dagi AB092709 This paper 3 Cy. cribratus ispirensis Turkey: Col d’Ovit, Dag (2500 m) AB092710 ” 4 Cy. porrectangulus porrectangulus Turkey: Yayladere, Kostandag, Yagmurdere (1900 m) AB017462 Imura et al. 1998a 5 Cy. cribratus pulumuricus Turkey: Mercon M.R.-S. Erzincan, Mt. Akbaba (2900 m) AB092711 This paper 6 Cy. porrectangulus porrectangulus Turkey: W. Giresun Mt. R. W. Erico Tepesi (1200 m) AB092712 ” 7 Cy. porrectangulus porrectangulus Turkey: Giresun Mt. R. Karagol Ridge (2500 m) AB092713 ” 8 Cy. cribratus cribratus Russia: Caucasus, Osetia, Mt. Khumaratkhok (2300 m) AB017461 Imura et al. 1998a 9 Cy. porrectangulus porrectangulus Turkey: W. Giresun Mts., Gonderic Tepesi (2000 m) AB092714 This paper 10 Cy. gemellatus gemellatus Iran: Tehrun, Gach-i-sar (2700 m) AB092715 ” Q- 1 Tomocarabus harmandi harmandi Japan: Tochigi, Nasu AB039810 Su et al. 2000 2 To. harmandi mizunumai Japan: Toyama, Arimine AB039813 ” R- 1 Pachycarabus koenigi koenigi Russia: Caucasus, Aibga Mts. (2400 m) AB092716 This paper 2 Pachyc. staehlini staehlini Russia: Caucasus, Osetia, Bokovoy Mts., AB092717 ” Sources of Riv. Liadon, Mt. Tepli (2700–3000 m) 3 & Pachyc. roseri ciftekopruleriensis Turkey: SE. slope of Mt. Karcal Dag (2500 m) AB092718 ” 4 S- 1 Euporocarabus hortensis hortensis Austria: Tirol, Innsbruck, Alpen Zoo AB017463 Imura et al. 1998a 2 Pachystus cavernosus variolatus Italy: Abruzzi, Gran Sasso, Campo Imperatore AB092719 This paper 3 Phricocarabus glabratus glabratus Austria: Tirol, Innsbruck, Stubaital, Volderau AB017466 Imura et al. 1998a 4 Ph. glabratus latior Italy: Piemonte, Cúneo, Canelli (450 m) AB017471 ” 5 Pachystus tamsi tamsi Iran~Azerbaidzhan, Savelan (2700 m) AB092720 This paper 6 Pachys. tamsi dashtensis Iran: Gilan, Damash~Kharpu, Cahor Mah.-yayla (1900 m) AB092721 ” T- 1 Meganebrius swatensis swatensis Pakistan: Swat, Utrot (2300–2600 m) AB092722 ” 2 Me. scheibei scheibei Pakistan: Dir, Lawarai Pass (3000–3100 m) AB092723 ” U1- 1 Titanocarabus titanus titanus China: Sichuan, Jiuzhaigou Xian, Jiuzhai Zhen (2100–2200 m) AB017474 Imura et al. 1998a 2 Ti. titanus titanus China: Sichuan, env. Nanping (1400 m) AB092724 This paper 3 Ti. titanus titanus China: Shaanxi, Liuba, Miaotaizi (1200 m) AB092725 ” 4 Ti. titanus titanus China: Shaanxi, Taibai Shan Mts. (2000 m) AB092726 ” 5 Ti. sui sui China: Beijing, Xiaolongmen AB017475 Imura et al. 1998a U2- 1 Piocarabus vladimirskyi vladimirskyi China: Beijing, Xiaolongmen (1120–110 m) AB092727 This paper 2 Pi. vladimirskyi vladimirskyi China: Qinghai, Qinghai Nan Shan Mts., 10km S. Heimahe AB092728 ” (3700m) U3- 1 Qinlingocarabus reitterianus reitteri- China: S Gansu, 40 km S Wudu, Tochizi (2300 m) AB092729 ” anus 2 Qi. kitawakianus kitawakianus China: S Shaanxi, 140 km S Xi’an, Pass 2100m, Huoditang-Xun- AB092730 ” yangba 3 Qi. nanwutai nanwutai China: S Shaanxi, 65 km S Xi’an Pass (2100 m) AB092731 ” 4 Qi. reitterianus reitterianus China: Gansu, S. of Lianghekou (2300 m) AB092732 ” 5 Qi. reitterianus reitterianus China: Shaanxi, Liuba, Gaoqiaopu (1400 m) AB092733 ” 6 Qi. reitterianus reitterianus China: Shaanxi, Liuba, Miaotaizi 1500–1600 mAB092734 ” 7-9 Rhigocarabus choui choui China: Shaanxi, Taibai Shan nat. park (3000–3200 m) AB092735 ” 10 Heptacarabus ohshimaianus China: Hubei, Shennongjia, Guanmen shan (1500 m) AB092736 ” 11 Qinlingocarabus blumenthaliellus China: Hubei, Shennongjia, Liaowangtai (2820 m) AB092737 ” V- 1 Leptocarabus kyushuensis Japan: Hiroshima, Kure D50356 Su et al. 1996a 2 Le. canaliculatus rufinus China: Liaoning, Dandong AB031474 Kim et al. 2000a W1- 1 Rhigocarabus handelmazzettii handel- China: Yunnan, NE. Zhongdian (4200–4400 m) AB050746 Su et al. 2001 mazzettii 2 Rh. handelmazzettii handelmazzettii China: Yunnan, NE. Zhongdian (3800–4000 m) AB092738 This paper

56 Z.-H. SU et al.

continued 3 Rh. handelmazzettii virginalis China: Yunnan, NE. Zhongdian (4300 m) AB092739 ” W2- 1 Rh. itzingeri rugulosior China: Yunnan, Dêqên, Mekong side of Hengduan Shan, AB092740 ” S. of Meilixue Shan (3800–4300 m) 2 Rh. itzingeri choguy China: Tibet, Zayü Co., upper Basin of AB092741 ” E. branch of Taron (Irrawaddy) Riv. (3800 m) 3 Rh. itzingeri choguy China: Tibet, Zayü Co., Luhit (Zayü), AB092742 ” Irrawaddy divide 18–25 km of Zayu town (4400–4600 m) W3- 1 Rh. roborowskii maniganggo China: Sichuan, Pass 20 km S. Qagca (4000 m) AB050747 Su et al. 2001 2 Rh. ladygini ladygini China: Qinghai, 300 km SW. Xining, Wenchuan env. (4000–4400 AB092743 This paper m) 3 Rh. ladygini ladygini China: Qinghai, 300 km SW. Xining, Ngola Pass (4500–4800 m) AB092743 ” 4 Rh. ladygini ladygini China: Qinghai, Pass N. of Huashixia (4600 m) AB092743 ” W4- 1 Rh. indigestus pseudoindigestus China: Sichuan, 20 km W. Yajiang, 150 km W. Kangding (4300- AB050748 Su et al. 2001 4600m) W5- 1 Rh. rhododendron rhododendron China: Yunnan, Baimaxue Shan, 35km SE. Dêqên (4300–4800 m) AB050749 ” W6- 1 Rh. zheduoshanensis zheduoshanensis China: Sichuan, W. of Kangding, Zheduo Pass (4300m) AB092744 This paper W7- 1 Leptocarabus yokoae nanjiangensis China: Sichuan, Nanjiang Xian, Mt. Guangwu Shan (1520 m) AB031426 Kim et al. 2000a 2 Le. yokoae nanjiangensis China: Sichuan, Nanjiang Xian, Micang Shan Mts., Daba (1350 m) AB031427 ” 3 Le. marcilhaci marcilhaci China: Gansu, 40km S. Wudu, Tochizi (2300 m) AB031425 ” 4 Le. yokoae yokoae China: Hubei, Shennongjia AB031428 ” W8- 1 Rhigocarabus laotse qinghaiensis China: Sichuan, Maniganggo W. env. (4100 m) AB050750 Su et al. 2001 W9- 1 Rh. cateniger cateniger China: Qinghai, Qinghai Nan Shan Mts., 10km S. Heimahe (3700 AB092745 This paper m) 2 Rh. cateniger cateniger China: Qinghai, Lajishan Mts., Lajishankou AB092746 ” 3 Rh. cateniger cateniger China: Qinghai, Laji Shan Mts., 50km S. Xining (3800–4000 m) AB092747 ” W10- 1 Rh. confucius kangdingensis China: Sichuan, Kangding, Pass Zheduo-shankou (3850 m) AB092748 ” 2 Rh. confucius kangdingensis China: Sichuan, Qianning~Danba, Pass 15km NE. Qianning AB092749 ” (3800m) 3 Rh. confucius kangdingensis China: Sichuan, Kangding, Pass Zheduo-shankou (3900 m) AB092750 ” 4 Rh. confucius kangdingensis China: Sichuan, Kangding, Pass Zheduo-shankou (4300 m) AB092751 ” W11- 1 Rh. pusio hylonomus China: Sichuan, Pass N. Zhangla (3800 m) AB050752 Su et al. 2001 2 Rh. pusio hylonomus China: Sichuan, Songpan Xian, Pass Gongga Ling (3500 m) AB017477 Imura et al. 1998a 3 Rh. pseudopusio pseudopusio China: Sichuan, NE. Sanggarmai (4500 m) AB050751 Su et al. 2001 4 Rh. buddaicus obenbergeri China: Qinghai, 25 km W. Tongren (3600–4150 m) AB092752 This paper 5 Rh. gigolo gigolo China: Sichuan, Zoige, Langmusi (Dogcanglhamo) (3600 m) AB092753 ” 6 Rh. buddaicus linxiaicus China: Gansu, Labrang, Val. E. of Ponggartang (3600 m, meadow) AB092754 ” 7 Rh. buddaicus linxiaicus China: Gansu, Labrang, Val. E. of Ponggartang (3000m, conifer- AB092754 ” ous forest) 8 Rh. buddaicus gansuicus China: Sichuan, Zoige, Langmusi (Dogcanglhamo) (4100 m) AB092755 ” W12- 1 Rh. maleki ssp. China: Sichuan, Shuajingsi, E. of Chiguguan (4300–4400 m) AB092756 ” W13- 1 Rh. latro qinlingensis China: Shaanxi, 65 km S. Xi’an (2100m) AB050753 Su et al. 2001 2 Rh. mikhaili mikhaili China: Gansu, 20 km N. Wenxian (3000m) AB050754 ” 3 Rh. latro minshanensis China: Sichuan, Col S. Jiuzhaigou (Nanping) (2500 m) AB050755 ” 4 Rh. latro minshanensis China: Sichuan, Jiuzhaigou Xian, Jiuzhaigou, Rizegou (3100 m) AB017476 Imura et al. 1998a X1- 1 Tomocarabus scabripennis ponticola Turkey: W. Giresun Mts., W. Gönderic Tepesi (2000 m) AB050757 Su et al. 2001 X2- 1 To. decolor decolor Russia: NW. Caucasus, Lagonaki, Krasnodar AB050758 ” 2 To. decolor decolor Russia: W. Caucasus, S. Slope of Aibga Mts. (2100–2200 m) AB050759 ” X3- 1 To. fraterculus jirisanensis S. Korea: Kyongsangnam-do, Chiri-san Mts. AB050760 ” 2 To. fraterculus odaesaniculus S. Korea: Kangwon-do, Odae-san Mts. AB050761 ” 3 To. fraterculus gaixianensis China: Liaoning, Dandong AB050762 ” X4- 1 To. convexus dilatatus Italy: Trentino-Alto Adige, Bolzano, Merano AB050764 ” Phylogeny of Latitarsi ground beetles 57

continued 2 To. convexus bucciarellii Italy: Piemonte, Cuneo, Cissone (Langhe) (600 m) AB092757 This paper 3 To. convexus convexus Slovakia: High Tatras, Tatra National Park, Stary Smokovec AB050765 Su et al. 2001 4 To. convexus acutangulus Turkey: 50km S. Trabzon, Zigana Pass (1900 m) AB092758 This paper X5- 1 To. opaculus opaculus Japan: Hokkaido, Nemuro AB039794 Su et al. 2000 2 To. opaculus opaculus Japan: Hokkaido, Imakane AB039801 ” 3 To. opaculus opaculus Japan: Hokkaido, Samani AB039803 ” 4 To. opaculus shirahatai Japan: Yamagata, Mt. Zao AB039808 ” X6- 1 To. marginalis marginalis Russia: Belgorod, near Borisovka forest AB050756 Su et al. 2001 X7- 1 To. shaheshang tianshuicus China: Gansu, 40 km S. Wudu, Tochizi (2300 m) AB092759 This paper 2 Scambocarabus kruberi gaiensis China: Liaoning, Dandong AB092760 ” X8- 1 Tanaocarabus sylvosus sylvosus USA: Wisconsin, Forest Co., 24 km E. of Eagle River AB092761 ” X9- 1 Tomocarabus loschnikovi laschnikovi Russia: Tuva, Akademika Obrucheva Mts., Koptu Riv. Val. (900 m) AB050767 Su et al. 2001 2 To. loschnikovi laschnikovi Russia: Krasnoyarsk, Sayanogorsk Dist., Cheremushki (500–800 m) AB050768 ” 3 To. loschnikovi laschnikovi Russia: Krasnoyarsk, Sayanogorsk Dist., Cheremushki, (2300 m) AB050769 ” X10- 1 To. slovtzovi slovtzovi Russia: Tuva, Akademika Obrucheva Mts., Sainak Pass (2100 m) AB050766 ” X11- 1 To. taedatus rainieri~agassii USA: Washington, Kittitas Co., Cle-Eluh AB050763 ” X12- 1 Tanaocarabus forreri ssp. Mexico: Durango, El Salto AB092762 This paper 2 Ta. forreri ssp. USA: Arizona, Cochise Co., Chiricahua Mts., Rustler Camp AB092763 ” 3 Ta. forreri ssp. Mexico: Chihuahua, Maderas AB092764 ” X13- 1 Semnocarabus bogdanowi bogdanowi China: Xinjiang, 100 km E. Narat (2500 m) AB092765 ” X14- 1 & Ulocarabus stschurovskii lineellus Tadzhikistan: Turkestan Mts., SW. Shakhristan Vill., left bank of AB092766 ” 2 Altykul Riv. (3000 m) X15- 1 Semnocarabus minimus minimus China: Xinjiang, SSE. of Takes, Narat Mts., Ural Riv. AB092767 ” X16- 1 Carpathophilus linnei linnei Slovakia: High Tatras, Tatras Netional Park AB017472 Imura et al. 1998a X17- 1 Semnocarabus transiliensis transiliensis Kazakhstan: Zailiiskii Alatau Mts., Bol. Alma-Atinka Val. (2500– AB092768 This paper 3300 m) 2 Se. regulus regulus Kirgiz: Terskei Ala-Too Mts., SE. Pokrovka Tshon Kizil Suu (2300 AB092769 ” m) 3 Se. regulus regulus Kirgiz: N. slope of Terskei Ala-Too Mts., Tastarata Mt., AB092770 ” S. Kadzhi-sai (2900m) 4 Se. regulus regulus Kirgiz: Baiduly Mts., Kichi-Kara-Kudzhur Riv. (3400m) AB092771 ” 5 Se. regulus regulus Kirgiz: Terskei Ala-Too Mts., Suiok Pass (4000 m) AB092772 ” 6 Se. regulus hauserianus Kirgiz: Terskei Ala-Too Mts., right border of Aksu Riv. (3400 m) AB092773 ” 7 Se. regulus hauserianus Kirgiz: Terskei Ala-Too Mts., Kok-kiia AB092774 ” 8 Se. regulus hauserianus Kirgiz: Terskei Ala-Too Mts., Turgen Riv., Kok-kiia Val. (3000 m) AB092775 ” 9 Se. erosus ssp. Kazakhstan: 20 km N. Bakanas (350 m) AB092776 ” 10 Se. erosus korolkowi Kazakhstan: Transili Alatau Mts., Kaskelen Val. (1200–1300 m) AB092777 ” 11 Se. erosus callosus Kazakhstan: Zailiiskii Alatau Mts., Bol. Alma-Atinskae Lake AB092778 ” (2500 m) 12 Se. erosus ssp. Kirgiz: Ala-Archa (1800 m) AB092779 ” 13 Se. erosus ssp. Kirgiz: Baiduly Mts., Kichi-Kara-Kudzhur Riv. (2700 m) AB092780 ” 14 Se. erosus ssp. Kirgiz: Dolon Pass, Kara-Kichi-Kudzhur (2700 m) AB092781 ” 15 & Se. erosus carbonicolor Kirgiz: Terskei Ala-Too Mts., S. Ak-sas, S. Temir-kanat (2400 m) AB092782 ” 16 17 Se. erosus ssp. Kirgiz: Atbashi Mts., near Bosogo Vil. (2600 m) AB092783 ” 18 Se. erosus ssp. Kazakhstan: Ketmen Mts., Tuiuk (2000 m) AB092784 ” 19 Se. erosus carbonicolor Kirgiz: Inyltshek Mts., near Maidaadyr (2800–3100 m) AB092785 ” * The specimen numbers correspond to those following the scientific names in the phylogenetic trees. those revised thereafter were adapted to those of the name in place of Carabus (s. lat.). results and discussion section. For convenience, the rou- The methods of DNA extraction, PCR amplification, tinely used subgeneric names were used as the generic sequencing and the construction of the phylogenetic trees 58 Z.-H. SU et al.

（a）�

（b）�

Fig. 1. Locality maps of the specimens used in this study. a, the map showing the localities of all over the specimens; b, the detailed map of Sichuan Province and the adjoining areas (Southwest China) showing the localities of the specimens of the lineage W. The locality numbers correspond to those shown in Table 1 and other figures. by using neighbor-joining (NJ) method, the unweighted division Latitarsi, together with a few representative spe- pair grouping method with arithmetic means (UPGMA) cies of other taxonomic divisions. The representative and the maximum parsimony (MP) method are according species in this division are illustrated in Fig. 3. On the to Su et al. (1996a, 1998) and Su et al. (2003). All these tree were recognized a considerable number of lineages methods yielded essentially the same topology unless oth- that emerged within a short period a little after or around erwise specified and only the NJ-trees were shown in this the time of radiation of the Carabina as judged by short paper. The dating was done, assuming that a 0.01 D branch lengths with low bootstrap values, so that their unit corresponds to 3.6 million years for the ND5 gene of branching order was unable to determine with certainty. the carabid beetles (Su et al., 1998, 2001). This was not because of saturation of nucleotide substitu- tions, since the actual percentage of substitutions was lin- early proportional to evolutionary distance, the value RESULTS corrected for multiple substitutions by Kimura’s method Fig. 2 shows the NJ-phylogenetic trees of almost all (1980). As compared with other divisions in which the genera that have been taxonomically classified into the members of each division were almost clearly clustered Phylogeny of Latitarsi ground beetles 59 together, the species belonging to the Latitarsi did not even weakly and indecisively clustered to the members of form a single cluster. Cavazzutiocarabus latreilleanus other divisions. In addition, the branching point of Auto- (lineage H) and Autocarabus cristoforii (lineage L) were carabus (lineage A), Mesocarabus (lineage B) and 60 Z.-H. SU et al.

Orinocarabus (lineage C) were as deep as those of other ent species in each genus were always clustered together taxonomic divisions such as Procrustimorphi (lineage D around the root of the lineage X in Fig. 2 upon various in Fig. 2a), Archicarabomorphi (E), Lipastromorphi (F), treatment, having given essentially the same topology. Digitulati (G), Lepidospinulati (lineage I), Crenolimbi (J), The details for each lineage are described as follows. Spinulati (K) and Arciferi (M), so that the lineages A, B and C would have no direct phylogenetic affinity with the Lineages A and L There existed two distinct lineages other members in the Latitarsi. In the other Latitarsi for the Autocarabus species, which revealed no direct phy- members, the lineages N to X were weakly clustered as logenetic linkages to each other on the tree (A and L in one group, respectively, but their topologies on the ND5 Fig. 2a). Their emergences were estimated to have tree were somewhat unstable upon replacement of an out- taken place around the same time of, or even earlier than, group, the use of the other methods of tree construction the radiation of the Carabina. The lineage A is divided such as the UPGMA or the MP, or an addition/removal of into two sublineages, A1 and A2, with a deep branching species. In Tomocarabus (except To. harmandi), Tanao- point. The A1 included a sole species, Autocarabus aura- carabus, Ulocarabus, Carpathophilus (sensu Imura et al., tus from eastern France and the A2 is constructed by sev- 1998a), Scambocarabus and Semnocarabus, the constitu- eral subspecies of Au. cancellatus from various localities Phylogeny of Latitarsi ground beetles 61

Fig. 2. NJ- phylogenetic trees of the mitochondrial ND5 gene of the subtribe Carabina. a, the overall tree; b and c, the detailed trees of the lineages W and X, respectively. The number in each branching point indicates the bootstrap percentage. The trees were out- group-rooted using the ND5 gene sequences of Cychrus morawitzi and Cy. thibetanus. Locality numbers after the scientific names correspond to those in Table l and Fig. 1. The capital letters show the name of each phylogenetic lineage. Thirteen sublineages of the lineage W are indicated on tree b, and seventeen sublineages of the lineage X are indicated on tree c. The scientific names of Latitarsi species are indicated in bold type on tree a. The lineages of other divisions are as follows: D, Procrustimorphi; E, Achicar- abomorphi; F, Lipastromorphi; G, Digitulati; I, Lepidospinulati; J, Crenolimbi; K, Spinulati; M, Arciferi. 62 Z.-H. SU et al.

Fig. 3. Photographs of representative species in the Latitarsi. The number of each photograph corresponds to that of Table 1 and other Figs. For the scientific names of the species, see Table 1 and phylogenetic trees. of Europe and western Russia. In the sublineage A2, specimens of subsp. graniger from western Russia. Mor- subspp. tuberculatus (western Russia), emarginatus phologically, they are characteristic in having metalli- (northwestern Italy) and carinatus (eastern France) were cally colored dorsal surface and marked preapical close to one another, and were considerably far from two emargination of the female elytra, which are rather Phylogeny of Latitarsi ground beetles 63

Fig. 4. NJ- phylogenetic tree of the mitochondrial ND5 gene of the “Oreocarabus” species with some other Latitarsi species. The number in each branching point indicates the bootstrap percentage. The tree was outgroup-rooted using the ND5 gene sequences of Cychrus morawitzi and Cy. thibetanus. The branches of “Oreocarabus” species are marked by bold lines. The phylogenetic lineages are indicated by the capital letters as same as those in Fig. 2. 64 Z.-H. SU et al. exceptional in the Latitarsi. On the other hand, (lineage S in Fig. 2a, and see below). endophallic morphology of these two species is similar, The Chinese “Oreocarabus”, with exception of Rhigo- and nobody has pointed out the fact that the Autocarabus carabus, formed a well-defined cluster on the ND5 tree species in lineage A occupies such an independent posi- (lineage U in Fig. 4), which is divided into three subclus- tion in the phylogeny of the Carabina. Indeed, the lin- ters. To the first one (sublineage U1), Titanocarabus eage A can be regarded not only as an independent genus titanus and Ti. sui belonged. The second subcluster (U2) but also as a distinct division on the ND5 tree. The lin- contained two specimens of Piocarabus vladimirskyi. eage L contained a single species, Autocarabus cristoforii, The third subcluster (U3) included all four species of Qin- which is a small-sized species having an external appear- lingocarabus (kitawakianus, reitterianus, nanwutai, ance considerably different from other Autocarabus spe- blumenthaliellus), Heptacarabus ohshimaianus, and cies, and is endemic to the Pyrenees of southwestern Rhigocarabus choui. As a whole, there was a good cor- Europe. This species is rather poor in its own character- relation between the morphology and the molecular phy- istic features, and yet its high phylogenetic independence logeny at a genus level. Two exceptions were that from other Autocarabus species is evident by the present Heptacarabus was completely included in the third sub- molecular phylogeny. cluster to which all the Qinlingocarabus species belonged. Rhigocarabus choui was clustered with the Lineage B Mesocarabus problematicus is widely dis- Qinlingocarabus species and was not included in the lin- tributed in central Europe and the adjacent islands, and eage W, to which all other Rhigocarabus species belonged was a sole constituent of the lineage B. The evolutionary (see below). This result shows that Heptacarabus, Qin- distance between the specimens from eastern France lingopcarabus and Rh. chuoi formed a single cluster. The (subsp. planiusculus) and that from northern Germany morphological reexamination of choui revealed that, in (subsp. harcyniae) was fairly large. Morphologically, spite of the close similarity of the external structure to Mesocarabus has no distinct features both in external and that of Rhigocarabus, endophallus of male genitalia was male genital structures separable from other Latitarsi undoubtedly of the Qinlingocarabus-type (Fig. 5, see Fig. members. However, the ND5 tree shows that it belongs 3). At a species level, Titanocarabus titanus and Ti. sui to an independent lineage, which may be regarded as a were intermingled in the tree, revealing small sequence distinct division. differences. The difference between Qinlingocarabus kitawakianus and Q. nanwutai was also very small. Lineages C, H, P, S, U, W13, and X16 These lineages As was pointed out by Imura et al. (1998a), two “Oreo- and sublineages had been taxonomically placed in one carabus” species, cribratus and porrectangulus, should be genus “Oreocarabus” (Imura 1996) consisting of five spe- placed in the genus Cytilocarabus. The sequence analy- cies-groups (Imura and Mizusawa, 1996). Four of them ses of additional specimens showed that Cy. cribratus and were previously analyzed for the ND5 sequence to know Cy. porrectangulus were not clearly separated on the phy- their phylogenetic relationships (Imura et al., 1998a). logenetic tree (lineage P in Fig. 4). Indeed, these two There were recognized eight independent lineages which “species” are morphologically very close with only a small were respectively treated as independent genera: difference in the shape of aedeagal apex of male Orinocarabus, Cavazzutiocarabus, Cytilocarabus, Eupor- genitalia. It would be adequate to regard these two as ocarabus, Phricocarabus, Titanocarabus, Rhigocarabus conspecific. “Oreocarabus” gemellatus from Iran is and Carpathophilus (Imura et al., 1998a). However, the unambiguously clustered with cribratus/porrectangulus phylogenetic positions of the remained species group “val- with a reasonable evolutionary distance. Thus, this Ira- adimirskyi” and most of the Chinese “Oreocarabus” spe- nian species should be considered as a member of the lin- cies are unknown yet, although Imura (1998) has eage P. rearranged them as follows on the basis of morphological characters with a reference of the molecular phylogenetic Lineages N and O Of the two species in the genus positions of Ti. sui and Rh. latro (Imura et al., 1998a): Eurycarabus, Eu. famini from North Africa was available Titanocarabus (titanus, sui); Qinlingocarabus (kitawaki- for the DNA analysis and formed an independent lineage anus, reitterianus, nanwutai, blumenthaliellus); Heptac- (lineage N). The genus Nesaeocarabus contains three arabus (ohshimaianus); Piocarabus (vladimirskyi); species, all endemic to the Canary Islands. One of them, Rhigocarabus (latro, qinlingensis, laotse, tewoenisis, Ne. abbreviatus (type species of Nesaeocarabus), was ana- mikhaili). As a part in this study, we analyzed more lyzed for the present study, which formed the lineage O, samples especially of the Chinese “Oreocarabus” species, and appeared to be related to the Eurycarabus lineage and reconstructed the phylogenetic tree for all the “Oreo- (N). This relationship between the two genera was also carabus” species (Fig. 4). Both the previous and present supported by other trees constructed by UPGMA and MP trees were essentially the same, except that Phricocara- method (not shown), although bootstrap values were not bus glabratus was clustered with the Pachystus species high. This result suggests a possibility that two genera Phylogeny of Latitarsi ground beetles 65

Fig. 5. Endophallus of male genitalia of several Latitarsi species. 1, Qinlingocarabus blumentaliellus; 2, Rhigocarabus choui; 3, Rh. confucius; 4, Rhigocarabus gigolo; 5, Rh. buddaicus.

would have been derived from the common ancestry, They are endemic to Caucasus including northeastern which had been isolated upon split of the African Conti- Turkey. Two species of the genus Meganebrius, swaten- nent and the Canary Islands. Morphologically, Eurycar- sis and scheibei, both from northern Pakistan, formed abus is a well-defined group by having a small head, a another cluster (lineage T). All the species in the lin- robust body, short antennae lacking the hairless ventral eages R and T bear dark and mat body surface and are depressions in the male, thick and characteristically similar in morphologies despite their phylogenetic inde- sculptured elytral surface and a unique membranous pro- pendences, suggesting that similar morphologies arose in jection on the ventral wall of the endophallus. Nesaeo- the two lineages in parallel. carabus is characterized by strongly polished body surface and completely degenerated ostium lobe of the Lineage S This lineage consisted of two major sublin- male genitalia. The most characteristic feature in the eages, which are represented by Euporocarabus and the endophallic morphology of Nesaeocarabus is the presence Pachystus-Phricocarabus complex, respectively. Pachys. of strongly sclerotized projection on the ventral wall, cavernosus from centro-eastern Italy was clearly clus- which is suggestive of the remote affinity with Eurycara- tered with Ph. glabratus from Austria, and Pachys. tamsi bus. from northern Iran was clustered with Ph. glabratus from northwestern Italy. The branching point between the Lineage Q This lineage contained only a single species, cavernosus-glabratus cluster and the glabratus-tamsi Tomocarabus harmandi, which is endemic to eastern cluster was rather deep with the emergence time about 20 Honshu, Japan. The geographic variations within the MYA (Su et al., 2001). On the other hand, Pachystus species have been investigated for details using the ND5 and Phricocarabus are morphologically classified into two gene sequences (Su et al., 2000). Fig. 2 showed that the distinct genera. The results may be interpreted in such phylogenetic position of To. harmandi is rather remote a way that the ancestor had been divided into two lin- from other Tomocarabus species. eages about 20 MYA, followed by a parallel evolution of in the respective lineages, probably by a discontinuous Lineages R and T Three species of Pachycarabus morphological change from Phricocarabus to Pachystus or (koenigi and staehrini from Caucasus, and roseri from vice versa. northeastern Turkey) formed one cluster (lineage R). 66 Z.-H. SU et al.

Lineage V This lineage was composed of the species accompanied by morphological changes. belonging to Leptocarabus (s. lat.) with exception of The sublineage W3 was composed of Rh. roborowskii two Chinese “Leptocarabus” species, yokoae and maniganggo from northwestern Sichuan and Rh. ladygini marcilhaci. Three main groups of Leptocarabus were from eastern Qinghai with a reasonably deep branching recognized by a molecular phylogenetic tree, if the Chi- point. nese species are included. They were the Japanese The sublineages W4-W6, W8 and W12 comprised only group, the continental group and the Chinese group (Kim a single species, respectively. W4 (Rh. indigestus from et al., 2000a). However, the Chinese group was not western Sichuan) might be remotely related to W5 (Rh. included in the lineage V and was a constituent of the lin- rhododendron from northwestern Yunnan). eage W (see below). The results confirmed the previous The sublineage W7 contained two “Leptocarabus” spe- analysis using mitochondrial and nuclear DNAs (Su et cies, yokoae and marcilhaci from centro-southern China, al., 2001). in the nearby regions of which some Rhigocarabus species are distributed. These two species are morphologically Lineage W The species in the genus Rhigocarabus very different from other Rhigocarabus species, and are were the main constituents of this lineage, with inclusion quite similar to some Japanese Leptocarabus species (see of two Chinese “Leptocarabus” species (Su et al., 2001; see Su et al., 2001). We have interpreted the results by above). Rhigocarabus contains 28 species and a number assuming that the Chinese and the Japanese Leptocara- of subspecies (Brˇezina, 1999). Most of them are distrib- bus evolved in parallel from two distinct ancestors (Su et uted in the high mountainous areas in southwestern al., 2001). Alternatively, it is possible that the Leptocar- China, and reveal similar external morphologies so as to abus ancestor was divided into three main groups, and make their classification rather difficult. In this study, then the Rhigocarabus species branched off from the Chi- 32 specimens comprising 19 species from various locali- nese Leptocarabus group with rapid morphological ties were examined for the ND5 sequences (Table 1, Fig. changes, and radiated. 1b). As shown in Figs. 2a and b, the Rhigocarabus spe- The sublineage W8 contained only one species Rh. cies were clustered well with a high bootstrap value, and laotse (subsp. qinghaiensis) from northwestern Sichuan. the Chinese “Leptocarabus“ species, yokoae and marcil- The sublineages W9 and W10 constituted three speci- haci, fell within this lineage. Thirteen sublineages were mens of Rh. cateniger from eastern Qinghai, and several recognized in this lineage, and eight of them respectively specimens of Rh. confucius from centro-western Sichuan, consisted of only a single species (Fig. 2b). The branch- respectively. The evolutionary distances between these ing order of the sublineages was unable to determine with species were small or almost null. certainty because of the short branching lengths with low The sublineage W11 was composed of four species, Rh. bootstrap values. In other words, a considerable number pusio (subsp. hylonomus from northern Sichuan), Rh. of sublineages would have radiated within a short time pseudopusio (from northern Sichuan), Rh. buddaicus about 30 MYA. We settled 13 sublineages, W1 through (subspp. obenbergeri from eastern Qinghai, linxiaicus and W13, for the sake of explanation. gansuicus from southern Gansu), and Rh. gigolo (south- The sublineage W1 contained two subspecies of Rh. ern Gansu). Rh. pusio and Rh. pseudopusio formed inde- handelmazzettii from northwestern Yunnan. They were pendent lines, respectively, and were distinctly separated phylogenetically close to, but morphologically distinguish- from Rh. buddaicus and Rh. gigolo on the tree, while the able from, one another. Note that one specimen (W1-2) evolutionary distance between Rh. buddaicus and Rh. of handelmazzettii s. str. from the lower altitudinal area gigolo was almost null. Morphologically, Rh. gigolo is (3800–4000 m) was closer to morphologically different one of the most specialized species having a peculiarly subspecies virginalis than to another specimen (W1-1) of sclerotized ostium lobe on the membranous preostium of handelmazzettii s. str. from the higher altitudinal area male genital organ, and is readily discriminated not only (4200–4400 m), suggesting that phylogeny of these two from all the subspecies of Rh. buddaicus but also from subspecies does not reflect the morphological difference, any other Rhigocarabus species (Fig. 5). These facts and subsp. virginalis branched off recently from one of suggest that a rapid morphological change took place the lines of handelmazzettii s. str. quite recently as in the case of W1. W11 was remotely The sublineage W2 included three specimens of Rh. itz- clustered with W10 (Rh. confucius) with a higher boot- ingeri, one (subsp. rugulosior) from northwestern Yunnan strap value, suggesting that W10 and W11 share the com- and two (subsp. choguy) from southeastern Tibet mon ancestry around the time of the Rhigocarabus (Xizang). The evolutionary distance between the Yun- radiation. nan specimen and the Tibet specimens was considerably The sublineage W12 contained Rh. maleki from centro- large, although these two populations show at most sub- northern Sichuan. specific morphological differentiation. The result sug- The sublineage W13 was composed of three species, Rh. gests that the geographical divergence is not always qinlingensis from southern Shaanxi, Rh. mikhaili from Phylogeny of Latitarsi ground beetles 67 southern Gansu, and Rh. latro from northern Sichuan. Rh. mikhaili was remotely related to Rh. latro, with Rh. qinlingensis as their outgroup. The three species in this sublineage and Rh. laotse (W8) had been placed in the latro species-group of the genus Oreocarabus (Imura and Mizusawa, 1996). The previous phylogenetic study on the “Oreocarabus” complex made it clear that latro should be placed to the genus Rhigocarabus and is not a member of Oreocarabus (Imura et al., 1998a). Other three spe- cies (laotse, qinlingensis and mikhaili) analyzed in this study also fell out in W13 of Rhigocarabus (Fig. 2b, see Fig. 6). As shown in Fig. 1b, not only the distribution range in the lineage W is narrowly restricted to southwestern China, but also each sublineage in W has its own habitat, strictly isolated from those of the other sublineages in most cases. This suggests, despite the long evolutionary history of this lineage, the members in each sublineage did not much expand their distribution after isolation, presumably because of their poor ability of immigration.

Lineage X The species morphologically classified into the genera Tomocarabus, Scambocarabus, Tanaocarabus, Ulocarabus, Carpathophilus and Semnocarabus (all established by Reitter) fell out in the lineage X with an exception of Tomocarabus harmandi (the loschnikovi group in Tomocarabus), which formed an independent lineage Q (see above). The branching order in X was ambiguous, as they were supported by low bootstrap value (see Fig. 2). Despite the low bootstrap value sup- porting the lineage X, the members in this lineage are morphologically similar and are always clustered together in the trees constructed by other methods, replacement of outgroups, and addition or deletion of the species. By the above reasons, we tentatively consider that the lineage X is a monophyletic. A detailed phylogenetic tree of the lineage X is shown in Fig. 2c. There were considerable inconsistencies between the morphological classification and molecular phylogeny. The species placed in the same genus did not form a single group; morphologically defined genus was not necessarily monophyletic. This situation was espe- Fig. 6. Comparison between morphological classification and cially apparent for the species of Tomocarabus (sensu molecular phylogeny of Latitarsi. The (sub)lineages estimated Imura & Mizusawa, 1996), which appeared in many dis- by ND5 trees are shown on the left, and the morphological gen- tinct clusters. Morphologically, Tomocarabus is divided era including some species groups are indicated on the into four species-groups, namely the convexus-group, the right. The genera marked by a star had been taxonomically placed in one genus “Oreocarabus” (Imura, 1996). marginalis-group, the loschnikovi-group and the taedatus- group (see Fig. 6). Of these, the marginalis group and the taedatus group correspond to X6 and X11, sublineages, X1 (scabripennis), X2 (decolor) and X4 respectively. The members of X6 are distributed in (convexus). X4 contained four subspecies of To. con- Eastern Europe through western Russia, and those of vexus, with comparatively large evolutionary distances X11 are endemic to North America. The remaining two between them. The X4 was further divided into two geo- groups were further divided into three to six sublineages graphic groups, one containing the specimens from Italy on the ND5 tree, and were apparently polyphyletic. The (subspp. dilatatus and bucciarellii) and another consist- species in the convexus group fell out in three different ing of those from Slovakia (nominotypical convexus) and 68 Z.-H. SU et al. northeastern Turkey (subsp. acutangulus). The sepa- two subspecies of Se. regulus (lutshniki and nominotypi- ration of the two groups would have been caused by geo- cal regulus) from the Terskei Ala-Toos. X17b was com- graphic isolation upon upheaval of the Alps about 20 posed of Se. regulus regulus from the eastern part of the MYA (Su et al., 2001). Although classified into the same Terskei Ala-Toos and several subspecies of Se. erosus group as that of To. convexus in morphology, To. scabrip- from southeastern Kazakhstan and northeastern Kirgiz. ennis formed an independent sublineage (X1). To. Note that Se. regulus appeared in two different subclus- decolor, which has a sclerotized projection on the ventral ters, and one of them was phylogenetically related to wall of the endophallus, also formed a distinct sublineage Se. erosus more than to the same species in another (X2). cluster. It is possible that the Se. regulus-like beetle is The species belonging to the loschnikovi group scat- the ancestral form of the X17 sublineage, from which Se. tered in the lineage Q and five different sublineages in erosus branched off within X17b. the lineage X, namely X3 (fraterculus), X5 (opaculus), X7 The remaining two genera in the lineage X, Ulocarabus (shaheshang), X9 (loschnikovi) and X10 (slovtzovi). The and Carpathophilus, formed two distinct sublineages lineage Q was represented only by To. harmandi, which (X14 and X16), which appeared to have little relations to is endemic to Japan (see above). To. opaculus is a sole the genus Semnocarabus. component of the sublineage X5, and is distributed in Japan, Sakhalin and the Kurils (see Su et al., 2000). The DISCUSSION sublineage X2 was composed of three subspecies of To. fraterculus from the Korean Peninsula and Northeast Comparisons of molecular phylogeny and taxon- China (Liaoning). Morphological differences among omy The Latitarsi was proposed by Imura (1996) as one these three subspecies are small, and yet their branching of the higher taxa of the subtribe Carabina. Although points were considerably deep. The sublineage X7 was originally established as one of five subdivisions in the composed of two Chinese species placed in two different Multistriati, the Latitarsi has been regarded as a distinct genera, Tomocarabus shaheshang from southern Gansu division by Imura et al. (1998b). This division is classi- and Scambocarabus kruberi from eastern Liaoning. fied into 17 (sub)genera (Imura, 1996), and some of them Although the branching point between them was very are further divided into several species-groups (Imura & deep, the cluster was supported by a high bootstrap value Mizusawa, 1996). of 74% (Fig. 2c). The molecular phylogeny suggests that at least 16 lin- The genus Tanaocarabus comprises three species eages, which have been morphologically placed in the endemic to North America, and two of them, sylvosus and Latitarsi, emerged at about the same time of the Cara- forreri, were available for the DNA analyses. Basic mor- bina radiation together with other divisions. Therefore, phological structure is very similar in both the species, these “Latitarsi” lineages may be regarded as separate and yet they belong to two independent sublineages (X8 divisions, respectively, especially in the case of the lin- and X12). This suggests that they arose in parallel or eage A (Autocarabus). The rest of the lineages, for exam- their morphology did not change much after their separa- ple, B, C, H and L may also be treated as the independent tion from the common ancestor. In X12 (Ta. forreri), the divisions. However, it is possible that at least some of evolutionary distance between a specimen from Durango these lineages had the common ancestry at the very (centro-western Mexico) and two specimens from Arizona beginning of their emergence. Since this possibility can- (southwestern USA) and Chihuahua (northern Mexico) not be verified by the present analyses, we have conven- was relatively large, while that of the latter two was tionally treated all the lineages as belonging to the small. Latitarsi without proposing new divisions to them. In The genus Semnocarabus is composed of 8 species any way, there exist considerable discrepancies between (Brˇezina, 1999), all endemic to the Tian Shan Mountains classification by morphology and molecular phylogeny, as in Central Asia. Five species were available for the shown in Fig. 6. A new classification scheme of the Lati- present DNA analyses. Morphologically, all the species tarsi based on the molecular phylogeny presented in this in this genus are similar to one another, and yet they fell paper will be published elsewhere by Imura as one of the out in three different phylogenetic lines on the ND5 possible taxonomic systems. tree. Three species, transiliensis, regulus and erosus, were included in the main sublineage X17, and two spe- Silent evolution A number of the genera in the Lati- cies, bogdanowi and minimus, both from Xinjiang of tarsi emerged within a short time a little after or around northwestern China, appeared so as to form distinct sub- the radiation of the Carabina 50–40 MYA (Su et al., lineages X13 and X15, respectively, separated from the 1996b; Imura et al., 1998a, b; Su et al., 2001), followed by main sublineage X17. X17 was further separated into at the secondary radiation of a number of species in the lin- least two clusters, X17a and X17b. The cluster X17a eages W and X about 30 MYA, and so on. This would contained Se. transiliensis from the Zailiiskii Alataus and imply that the radiation of the Latitarsi took place with Phylogeny of Latitarsi ground beetles 69 various scales and periods as has been recognized in carabus spp., and have been believed to be the closest to many other Carabina divisions such as the Digitulati, the Leptocarabus (s. str.) of the Japanese Islands, above all to Procrustimorphi, etc. (Su et al., 2001). Morphological Le. kyushuensis. However, the Chinese species are phy- diversifications of not a few species in the Latitarsi are logenetically independent from any other Leptocarabus rather poor, and yet they radiated in the early stage of the species from Japan, Korea and eastern Eurasia. In Carabina evolution. For example, the external morphol- other words, the Leptocarabus-like morphology appears ogy of almost all the species is alike throughout Tomocar- in the Rhigocarabus lineage in parallel with the authentic abus and its related genera (the lineage X). Leptocarabus lineage (see Su et. al., 2001). As noted in Many phylogenetic (sub) lineages treated in this study the results, an alternative interpretation would be that are composed of only a single species or at most two to the ancestor of Leptocarabus species first divided into two three allied species. It is remarkable that no other spe- lineages, the Chinese Leptocarabus lineage (yokoae and cies branched off from the lineages of Tomocarabus har- marcilhaci) and Continental/Japanese lineage, and then mandi (Q), To. fraterculus (X3), To. convexus (X4), To. Rhigocarabus branched off from the Chinese Leptocara- opaculus (X5), To. marginalis (X6), To. loschnikovi (X9), bus lineage presumably with discontinuous morphological or else, despite that initiation of their ND5 sequence change. diversification took place about 30 MYA. The main Rhigocarabus choui, which has a general appearance cause of the ND5 diversification in each lineage would hardly distinguishable from other Rhigocarabus species, most probably be geographic isolation resulted by forma- and yet its male genitalia is of the Qinlingocarabus- tion of the various barriers such as mountains, deserts, type. Phylogenetically it is placed in the Qinlingocara- tectonic lines, straits, rivers, etc., since the apterous bus cluster (the lineage U). This fact suggests that the ground beetles such as the Carabina cannot cross these occurrence of morphological transformation from the Qin- barriers. This is best exemplified by two populations of lingocarabus type to the Rhigocarabus type without To. convexus bordered by the Alps, which were completed accompanied change in the structure of male genitalia. about 20 MYA. The evolutionary distance between them The emergence of Rhigocarabus gigolo (W11) or Semno- is surprisingly large in spite of almost no accompanied carabus erosus (X17b) from another species with accom- morphological differences. panied morphological changes would also be the cases in A similar silent evolution is also observed in the Rhig- which recent discontinuous morphological changes took ocarabus species (lineage W), which inhabit the high place. A parallel evolution of Phricocarabus and Pachys- mountains of southwestern China, with the distributional tus in two different lines would be also an example of dis- range of each species usually restricted. Despite the continuous morphological change. Such a discontinuous long evolutionary histories, differences in the external evolution, sometimes accompanied by parallel evolution, morphology among various species of Rhigocarabus are often occurred during the Carabina evolution (Su et al., by no means conspicuous. 1996c; for review, see Osawa et al., 1999). The facts enumerated in this section imply that after emergence of various geographic isolates, only molecular We are grateful to the following colleagues for supplying the clock has been working with little morphological differen- specimens used in this study: I. A. Belousov (St. Petersburg, Rus- tiation for their long evolutionary histories, suggesting sia), E. Van Den Berghe (Seattle, USA), B. Brezina (Prague, that geographic isolation per se is not the sole cause of the Czech), R. Businsky & L. Businska (Prague, Czech), P. Cavazzuti (Pagno, Italy), M. L. Danilevsky (Moscow, Russia), R. Davidson speciation. In other words, the extent of morphological (Pittsburgh, USA), W. Heinz (Schwanfeld, Germany), I. I. Kabak diversification did not run in parallel with the evolution- (Almaty, Kazakhstan), Nobuo & Taeko Kashiwai (Kawasaki, ary distance, i.e., the approximate time elapsed after Japan), D. Kral (Prague, Czech), B. Lassalle (Boissy les Perche, emergence of the respective species. This stands a sharp France), J. A. Marrero A. (Tenerife, Spain), S. Murzin (Moscow, contrast to the situation, for example, within the division Russia), D. Obydov (Moscow, Russia), P. A. Oudovichenko (Mos- cow, Russia), A. Plutenko (Smolensk, Russia), P. Ratti (Biella, Procrustimorphi, where tremendous morphological diver- Italy), M. Satô (Nagoya, Japan), K. Staven (Lengede, Germany) sification occurred (Su et al., 2001). Such a silent mor- and J. Turna (Kostelec na Hane, Czech). Thanks are also due phological evolution is not rare in evolution of other to Hideko Kanda (formally Tanaka) for skillful technical carabid beetles as well, such as Apotomopterus sauteri assistance. 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