Resurrection of Rabdophaga salicivora Shinji (Diptera: Cecidomyiidae), a Japanese gall midge formerly misidentified as a North American species, Rabdophaga rigidae (Osten Sacken), with observations on the phylogenetic relationships of its populations in Japan and the Russian Far East

Collection and preservation of specimens

Galls of Japanese R. rigidae on Salix spp., North American R. rigidae on Salix lasiolepis Bentham, Russian R. salicivora on Salix spp., Rabdophaga rosaria (Loew, 1850) on Salix purpurea Linnaeus, and Rabdophaga terminalis (Loew, 1850) on Salix fragilis Linnaeus were collected from various localities in Japan, Poland, Russia, UK, and the USA between 2000 and 2003 (Table 1). The collection sites of Russian R. salicivora and Japanese R. rigidae used for molecular analysis are shown in Figure 1.

Some of the galls were dissected under a stereoscopic microscope to obtain larval and pupal specimens. When some of the dissected galls contained mature larvae or pupae, the rest of the collected galls were kept in plastic cylinders (30 cm in diameter, 37 cm in height) to obtain adults and pupal exuviae. All specimens collected in the present study were preserved in 75% ethanol for morphological examination and 99.5% acetone for molecular phylogenetic analysis.

All specimens used in the present study are kept in the collection of the Entomological Laboratory, Faculty of Agriculture, Kyushu University, Japan.

Morphological studies and terminology

For microscopic study, some of the ethanol-preserved specimens were mounted on slides in Canada balsam using ethanol and xylene. Drawings were made with the aid of a drawing tube. Adult morphological terminology follows usage in Gagné (1981), except for thoracic setae and the mediobasal lobe of the male genitalia, for which terminology follows usage in Yukawa and Ohsaki (1988) and Nijveldt and Yukawa (1982), respectively. Morphological terminology of larvae generally follows usage in Möhn (1955; originally written in German) that was translated into English in Yukawa (1971), and the terminology for pupae follows that in Gagné (1994).

In order to compare larvae, pupae, and adults of Japanese R. rigidae with those of North American R. rigidae, we borrowed slide-mounted specimens of 15 males, eight females, one pupa, and one larva of North American R. rigidae from the National Museum of Natural History, Smithsonian Institutions.

Molecular phylogenetic analysis

Acetone-preserved specimens of some Rabdophaga gall midges were used for molecular phylogenetic analysis (Table 1). Asphondylia yushimai Yukawa and Uechi 2003 was used as an outgroup species for the analysis. For every individual, total DNA was extracted from the whole body with the DNeasy tissue kit (Qiagen, Japan) following the manufacturer’s instructions. A region of the cytochrome oxidase subunit I (COI) gene of mtDNA was amplified, purified, sequenced, and electrophoresed following the methods described by Yukawa et al. (2003). The primers used for the amplification were as follows: forward, 5′-GGATCACCTGATATAGCAT TCCC-3′ (COIS) and reverse, 5′-CCCGGTAAAAT TAAAATATAAACTTC-3′ (COIA). This primer set has been effectively used for the analysis of intra- and interspecific variations in Cecidomyiidae (e.g. Uechi et al. 2003; Yukawa et al. 2003; Tokuda et al. 2004; Uechi et al. 2004). The nucleotide sequence data reported in this paper have been deposited in the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank nucleotide sequence databases with the accession numbers shown in Table 1.

The sequence data were analyzed with the neighbor-joining method using the software package phylip Version 3.573c (Felsenstein 1993). Evolutionary distances were calculated using Kimura’s two-parameter method (Kimura 1980). The resulting trees were subject to bootstrap analysis (Efron 1982; Felsenstein 1985) with 1000 replications for the neighbor-joining cladogram.

Morphological studies

Morphological studies of North American R. rigidae and Japanese R. rigidae, together with molecular analysis of North American R. rigidae, Japanese R. rigidae, and Russian R. salicivora, revealed that the taxonomic position of Japanese R. rigidae should be revised to Rabdophaga salicivora Shinji, 1938 (Shinji 1938a), which is redescribed as follows, together with the designation of a neotype. Hereafter, we use the name, Japanese R. salicivora instead of Japanese R. rigidae.

Rabdophaga salicivora Shinji sp. rev.

Rhabdophaga salicivora Shinji (1938): Shinji (1938a).

Rhabdophaga salicivora Shinji: Shinji (1938b, 1939, 1944); Monzen (1955); Kovalev (1967); Yukawa (1971).

(= Rabdophaga rigidae (Osten Sacken): Nijveldt & Yukawa 1982, misidentification).

Male. Eye bridge five or six facets long medially; frontoclypeal setae dense, 23–60 in number. Palpus four-segmented; second palpal segment 0.9–2.1 times as long as first; third 0.9–1.3 times as long as second; fourth 1.2–2.1 times as long as third. Antenna: scape ventrolaterally with 6–23 setae; pedicel ventrally and dorsally with 8–29 setae; with 18–22 flagellomeres; basal enlargement of fifth flagellomere 0.9–1.4 times as long as wide; terminal flagellomere relatively small, rounded apically. Wing length 3.2–5.2 mm, 2.4–2.7 times as long as wide; R5 joining costa near or at wing apex. Legs densely covered with blackish brown hairs; tarsal claws of all legs toothed (Fig. 2); empodia as long as claws; length of respective segments as in Table 2. Abdominal tergites I–VI rectangular, wider than long, with caudal rows of setae and one pair of anterior trichoid sensilla; tergite VII narrower than preceding, the vestiture similarly arranged; tergite VIII not sclerotized, bare and with a pair of anterior trichoid sensilla. Abdominal sternites II–VII rectangular, wider than long, with caudal rows of setae, with scattered setae elsewhere; sternite VIII not sclerotized, with scattered setae elsewhere. Genitalia (Fig. 4) yellowish brown; cerci setose, each large, rounded apically; hypoproct nearly as long as and distinctly narrower than cerci, the apex concave; gonostylus weakly arched, tapering distally, with strong apical claw; inner angle of gonocoxite ventrally developed into a setose lobe provided with a few protuberances ventrodistally; aedeagus longer than hypoproct, cylindrical, distally narrower, apically truncated or slightly rounded. See Table 2 for detailed data of setal counts and measurements.

Table 2. Rabdophaga salicivora Shinji: frontoclypeal and mesepimeral setal counts, and measurements of wing, fifth flagellomere and legs

	Male			Female
	n	Mean ± SD	(Range)	n	Mean ± SD	(Range)
Setae
Frontoclypeal	5	43.2 ± 15.8	(23–60)	3	54.0 ± 18.5	(40–75)
Mesepimeral	7	51.0 ± 13.1	(32–69)	6	76.2 ± 11.2	(63–93)
Wing
Length (mm)	7	4.4 ± 0.6	(3.2–5.0)	8	4.9 ± 0.2	(4.7–5.2)
Width (mm)	8	1.8 ± 0.2	(1.4–2.1)	8	1.9 ± 0.2	(1.8–2.4)
Length/width	7	2.5 ± 0.1	(2.4–2.7)	8	2.6 ± 0.2	(2.1–2.8)
Palpal segment length (µm)
First	7	65.7 ± 17.6	(34.2–83.1)	6	66.0 ± 14.1	(39.1–78.1)
Second	7	69.9 ± 8.8	(53.8–78.2)	7	69.2 ± 7.7	(63.6–83.1)
Third	5	78.2 ± 12.9	(63.6–97.8)	5	84.1 ± 14.1	(73.4–102.7)
Fourth	5	106.6 ± 25.7	(88.0–151.6)	5	106.6 ± 13.6	(88.0–122.3)
Fifth flagellomere
Length of basal enlargement (µm)	8	80.7 ± 8.3	(73.4–97.8)	8	81.3 ± 5.2	(73.4–88.0)
Width of basal enlargement (µm)	8	71.5 ± 8.2	(58.7–83.1)	8	60.5 ± 6.4	(48.9–68.5)
Length of stem (µm)	8	58.7 ± 5.2	(48.9–63.6)	8	8.0 ± 2.5	(4.9–9.8)
Foreleg (mm)
Fe	8	1.6 ± 0.2	(1.3–1.8)	7	1.6 ± 0.2	(1.4–1.8)
Ti	8	1.6 ± 0.2	(1.1–1.7)	7	1.6 ± 0.1	(1.4–1.7)
T2	8	1.6 ± 0.2	(1.2–1.8)	6	1.5 ± 0.1	(1.4–1.6)
T3	8	0.7 ± 0.1	(0.5–0.7)	6	0.6 ± 0.1	(0.5–0.6)
T4	8	0.4 ± 0.1	(0.3–0.5)	5	0.4 ± 0.0	(0.3–0.4)
T5	7	0.2 ± 0.0	(0.2–0.3)	5	0.2 ± 0.0	(0.2–0.2)
Midleg (mm)
Fe	8	1.7 ± 0.2	(1.2–1.8)	6	1.7 ± 0.1	(1.5–1.8)
Ti	8	1.6 ± 0.2	(1.2–1.8)	7	1.6 ± 0.2	(1.4–1.8)
T2	8	1.6 ± 0.2	(1.1–1.7)	6	1.4 ± 0.1	(1.3–1.5)
T3	8	0.7 ± 0.1	(0.4–0.7)	6	0.6 ± 0.0	(0.5–0.6)
T4	8	0.4 ± 0.1	(0.3–0.5)	6	0.4 ± 0.0	(0.3–0.4)
T5	8	0.2 ± 0.0	(0.1–0.3)	6	0.2 ± 0.0	(0.2–0.3)
Hindleg (mm)
Fe	8	1.8 ± 0.2	(1.5–2.0)	7	1.9 ± 0.1	(1.8–2.1)
Ti	8	1.9 ± 0.3	(1.4–2.2)	7	2.0 ± 0.1	(1.8–2.1)
T2	5	2.1 ± 0.2	(1.8–2.3)	7	1.9 ± 0.2	(1.7–2.2)
T3	5	1.0 ± 0.1	(0.9–1.0)	7	0.8 ± 0.1	(0.7–0.9)
T4	5	0.6 ± 0.1	(0.5–0.6)	7	0.5 ± 0.1	(0.5–0.6)
T5	5	0.3 ± 0.0	(0.3–0.3)	7	0.2 ± 0.0	(0.2–0.3)

Female. Eye bridge and palpal segments as in male. Antenna with 20–24 flagellomeres; basal enlargement of fifth flagellomere 1.1–1.8 times as long as wide. Abdominal tergites I–VI rectangular, wider than long, with caudal rows of setae and a pair of anterior trichoid sensilla; tergite VII narrower than preceding, the vestiture similarly arranged; tergite VIII not sclerotized, with caudal rows of setae and one pair of anterior trichoid sensilla. Abdominal sternites II–VI rectangular, wider than long, with rows of caudal setae, with scattered setae elsewhere; sternite VII rectangular, narrower than preceding, with scattered setae elsewhere and one pair of anterior trichoid sensilla; sternite VIII not sclerotized, bare. Ovipositor yellowish brown, elongate, 419.3–516.8 µm long, not sclerotized; cerci elongated, entire (Fig. 6). See Table 2 for detailed data of setal counts and measurements.

Gall. Subglobular or spindle-shaped, woody swelling on the twig (Fig. 11); color green, shading to brown; length 8–20 mm, diameter 5–18 mm; monothalmous (with single larval chamber). Japanese name of the gall: “Yanagi-zuifushi” (Monzen 1929; Monzen 1955; Yukawa 1976), “Yanagi-eda-tamafushi” (Shinji 1944), “Yanagi-eda-maruzuifushi” (Yukawa and Masuda 1996).

Molecular phylogenetic analyses

The amplified mtCOI gene fragment was 439 bp long. This region corresponded to bases 1752–2190 of the genome of Drosophila yakuba Burla (Diptera: Drosophilidae) (Clary & Wolstenholme 1985). The monophyly of the clade including Russian R. salicivora from Khanka Lake and Komissarovo, Russia and Japanese R. rigidae from Niigata, Aichi, and Miyazaki Prefectures, Japan (East Asian clade hereafter) was supported by a 90.9% bootstrap value (Fig. 12). The clade including North American R. rigidae individuals (North American clade hereafter) was more distant from the East Asian clade than from the clade including R. rosaria and R. terminalis individuals from Europe (Fig. 12). There were 51 (12.9% of 439 bp) to 71 bp (18.7%) differences (Table 3) and 8–13 differences in the 146 deduced amino acid residues between the East Asian and North American clades.

Several haplotypes were included in the East Asian clade. Individuals obtained from one locality formed one clade, except for those collected from Khanka Lake, which were distantly divided into two clades (Fig. 12). As for the Japanese individuals, those from Miyazaki Prefecture were closer to Russian R. salicivora individuals from Komissarovo or some of the Russian R. salicivora individuals from Khanka Lake than to Japanese R. salicivora from Aichi or Niigata Prefecture (Fig. 12).

Differences between North American R. rigidae and Japanese R. salicivora

We found two morphological differences between North American R. rigidae and Japanese R. salicivora with respect to the shape of the tarsal claws and the aedeagus of the male genitalia. North American R. rigidae has untoothed tarsal claws on all legs (Fig. 3) and an apically rounded aedeagus (Fig. 5), whereas Japanese R. salicivora has toothed tarsal claws on all legs (Fig. 2) and apically truncated or slightly rounded aedeagus (Fig. 4). The gall shapes of North American R. rigidae and Japanese R. salicivora are somewhat different. The former transforms the buds into subglobular to tapered galls (Gagné 1989), and the latter into smooth subglobular galls (Fig. 11). Molecular analysis indicated that North American R. rigidae and Japanese R. salicivora are included in two different clades: the North American and the East Asian clades (Fig. 12). In addition, three individuals of R. rosaria and R. terminalis collected from Europe were located between the North American and East Asian clades (Fig. 12). Based on these results, we conclude that the two species are quite distinct.

As a result, no known intercontinental biotic connections exist between North America and the Japanese Archipelago at the species level in gall-inducing cecidomyiids, except for the following species, which have been accidentally introduced into Japan: Contarinia maculipennis Felt and Obolodiplosis robiniae (Haldeman) (Gagné 1984, 2004; Tokuda et al. 2002; Duso & Skuhravá 2003; Kodoi et al. 2003; Uechi et al. 2005).

Derivation of Japanese R. salicivora

Molecular analysis indicates that Japanese and Russian R. salicivora are identical because the monophyly of the East Asian clade was supported by a high bootstrap value (90.9%; Fig. 12). Within the East Asian clade, Japanese R. salicivora individuals collected from Miyazaki Prefecture, southern Japan were closer to Russian R. salicivora individuals than those from Niigata and Aichi Prefectures, central Japan. In addition, Salix stem galls that are quite similar to those induced by R. salicivora have been recorded from the Korean Peninsula (Saitô 1932; Paik et al. 2004). These observations indicate that Japanese R. salicivora possibly came to the Japanese Archipelago through the Korean Peninsula when both areas were connected, and that it become established first in the southern parts of Japan. Then, it expanded its distribution range to the northern parts of Honshu, but did not reach Hokkaido (Yukawa & Masuda 1996), probably because of the existence of the Tsugaru Strait between Honshu and Hokkaido.