A new species of the genus Ambrostoma Motschulsky (Coleoptera, Chrysomelidae, Chrysomelinae) from South Korea, with larval descriptions and biological notes

Abstract Ambrostoma koreana sp. n. is described from South Korea. Detailed descriptions and illustrations of adult and all larval instars are provided and differences to closely related species are discussed. Ovoviviparity is reported for the first time in the genus Ambrostoma. Notes on distribution, host plant and biology are also provided.


introduction
The genus Ambrostoma was described by Motschulsky (1860) for three species from Russia (Amur region), China and Nepal, respectively, with A. quadriimpressum (Motschulsky, 1845) [= A. superbum (Thunberg, 1787)] as its type species. Since then, nine species have been described in East Asia and the Himalayas (Achard 1922, Chen 1934, 1936, Gressitt and Kimoto 1963, Wang 1992, Kimoto and Osawa 1995, Medvedev 2007. Chen (1936) divided the genus into two subgenera, Ambrostoma and Parambrostoma, based on elytral punctures, setae on elytral epipleura and length ratio of antennomeres, and later Wang and Chen (1981) raised these subgenera to the generic status. These two genera were recently revised to include fifteen species and the monophyly of both genera was well supported by morphological characters of adults (Ge et al. 2012). Ambrostoma is widely distributed in East Asia and comprises eight species. Members of the genus are characterized by a distinct transverse depression near the base of elytra, inner margin of elytral epipleura with setae along an entire length, procoxal cavities open posteriorly, metasternal process immarginate apically and tarsal claws simple. The larva are only known and for only one species of the genus: A. superbum which is a major pest in China, has been described by Medvedev and Voronova (1976). Ambrostoma larva is easily recognized among the subfamily Chrysomelinae by their orange stripes in live specimens, undeveloped tubercles, a dark pattern on the dorsum and black markings on the head and pronotum.
In 2006, the first author collected unusual Ambrostoma specimens, adults and larvae, on Namhaedo Island in South Korea. After a detailed examination we concluded that they belong to a new species described below. We compared the larval morphology of Ambrostoma to those of Parambrostoma. Notes on distribution, host plant, biology and occurrence of ovoviviparity are also provided.

Material and methods
The type specimens were deposited in the Department of Biodiversity and Evolutionary Taxonomy, University of Wrocław, Poland (DBET) and H.-W. Cho's private collection, Republic of Korea (HWC). Biological observations were made in April-May 2006, at the type locality and under laboratory conditions. Adults and larvae collected from the type locality were kept in plastic containers (10 cm diameter, 12 cm deep) with leaves of host plant and checked daily. All larval specimens used in the study were preserved in 70% ethanol. For morphological studies of minute structures, some larvae were dissected, cleared in 10% sodium hydroxide solution, rinsed in distilled water, and then mounted on slides with Swan's liquid (20 g distilled water, 15 g gum arabic, 60 g chlorhydrate, 3 g glucose, and 2 g glacial acetic acid). Genitalia were dissected from adult specimens softened in plastic containers with wet tissue paper for 12-24 hours. The aedeagus was softened in 10% sodium hydroxide solution for 6-12 hours and placed in distilled water. The careful insertion of a sharp-pointed thick nose hair and injection of 5% ethanol into the foramen of aedeagus were repeated until the internal sac was fully everted. After washing with absolute ethanol, the genitalia were preserved in a microvial with glycerin and pinned with the specimen. Descriptions and illustrations were prepared using Nikon SMZ800 and Nikon Eclipse E600 microscopes, each equipped with a drawing tube. Habitus images were taken by a Nikon Coolpix 4500 digital camera attached to a Nikon SMZ1500 microscope. The letters L, S and M in parentheses signify long, short, and micro setae, respectively. Adult (Figs 1-7, 19). Diagnosis. The new species is very similar in coloration to A. leigongshana Wang, but can be distinguished by the following characters: antennomere 3 much longer than 4 (equal in length in A. leigongshana); pronotum with moderately dense punctures in lateral depression (dense and coarse punctures in A. leigongshana); aedeagus widest at apical 1/5, thence narrowed with trapezoidal apex (elongate apically with rounded apex in A. leigongshana); spermatheca almost straight, curved at apex (strongly curved in A. leigongshana).

Ambrostoma koreana
Description. Holotype (Figs 1-2). Body length 11.7 mm, width 5.7 mm, strongly convex dorsally. Head emerald green to ultramarine with 2 pairs of small orange yellow markings on central part. Mouthparts midnight blue. Antennomeres 1-7 ultramarine, 8-11 black. Pronotum emerald green to ultramarine with a pair of large orange yellow markings. Scutellum ultramarine. Elytra ultramarine with a pair of orange yellow markings at base and 2 pairs of orange yellow longitudinal markings on centralposterior part, all markings surrounded by emerald green. Venter mainly ultramarine with greenish luster. Legs midnight blue.
Pronotum. Length 2.7 mm, width 5.4 mm. Lateral sides roundly widened anteriorly, widest at anterior 1/4. Anterior margin widely emarginated. Trichobothria on anterior and posterior angles. Disc with dense and small punctures, larger than those of head; hardly confluent large punctures in lateral longitudinal depression; interspaces with spare and minute punctures. Scutellum subtriangular, slightly wider than long, impunctate.
Elytra. Length 8.8 mm, width 6.8 mm. Sides moderately widened posteriorly and widest at posterior 2/5, thence rounded at apex. Disc with double irregular rows of punctures; transverse depression with large punctures, subequal to those of side of pronotum; interspaces with spare and minute punctures. Epipleuron flat, inner margin with micro setae along an entire length. Hind wing well developed.
Head. Vertex and temporal side with 30-32 pairs of long setae and 15-19 pairs of short setae. Frons with 16-19 pairs of long setae. Clypeus and labrum, each with 2 pairs of long setae. Mouthparts similar in shape and chaetotaxy to those of the last instar larva, except for stipes with 3 setae and postmentum with 3 pairs of setae.
Third instar larva. Description. Similar to the last instar larva except for following characters: body length 7.2-8.1 mm, width 2.8-3.8 mm, head width 1.90-2.05 mm (n = 3); dark patterns on dorsum much larger than those of the last instar larva. (Figs 8-14, 16, 18). Diagnosis. The last instar larva is easily distinguished from larva of A. superbum (Thunberg) in the following characters: pronotum with 3 black markings (2 black markings in A. superbum); dorsolateral posterior region of each abdominal segment without dark patterns (with dark patterns in A. superbum); width of peritreme less than half width of abdominal segment (equal to half width in A. superbum).
Etymology. This endemic species is named after the type locality, Korea.  Distribution. Specimens were collected in the southeastern part of the Korean Peninsula and on adjacent Namhaedo Island.
Biological notes. Overwintered adults were observed under bark or in fallen leaves near the base of trees in early April (Fig. 19). Adults and larvae fed on leaves of Zelkova serrata Makino (Ulmaceae) which is quite a common tree in Korea. Ulmus pumila Linnaeus (also Ulmaceae) was the only known host plant for three other Ambrostoma species (Yu et al. 1996). On 7 May 2006, a female laid 4 eggs that contained fully developed embryos in the laboratory. Four females were dissected shortly afterward, and 17-79 enclosed larvae were found in abdomen (Fig. 20). About 50 ovoviviparious species are actually known in 7 genera of Chrysomelinae (Bontems 1988, Bontems andLee 2008). Ovoviviparity is reported for the first time in the genus Ambrostoma.

Discussion
The Ambrostoma species has not been recorded in Korea since Chen (1936) mentioned A. superbum without any locality data and comments. Ambrostoma superbum is widely distributed in Southeastern Siberia, Eastern Mongolia, Northeastern China and probably North Korea (Kippenberg 2010, under name A. quadriimpressum quadriimpressum), whereas A. koreana is restricted to Namhaedo Island and Miryang, Gyeongnam Province, South Korea. The first instar larva of Ambrostoma was described for the first time. The second and third instar larvae are very similar in shape and coloration to the last instar larva, but the first instar larva distinctly differs in having more developed tubercles and long setae. The tubercular pattern of the first instar larva was similar to some larvae of the genus Chrysolina Motschulsky. Based on body shape, tubercular pattern and biology, Ambrostoma larva belongs to the generic group Chrysolina proposed by Kimoto (1962). The larva of Parambrostma mahesa (Hope) also belongs to the generic group Chrysolina (Takizawa 1989). The monophyly of both genera, Ambrostoma and Parambrostoma carried out by Ge et al. (2012), was supported by larval characters.
The mature larvae of these two genera are similar in having undeveloped dorsal tubercles, sclerotized platelets and sparse micro setae on dorsum. However, the considerable differences can be summarized as follows: dark patterns present on dorsum in Ambrostoma and absent in Parambrostoma; head and pronotum are pale coloured with black markings in Ambrostoma and entirely black in Parambrostoma; prothoracic and ventral tubercles are not scleotized in Ambrostoma, whereas they are well scleotized in Parambrostoma; basal tooth of tarsungulus in Ambrostoma is weakly developed while in Parambrostoma it is strongly developed.