Anopheles (Cellia) yaeyamaensis Somboon & Harbach, sp. n.

Anopheles formosaensis I of Miyasaki, 1902: 66 (Ishigaki Island).

Anopheles minimus of Bohart, 1959: 195 (Ishigaki and Iriomote Islands; L bionomics); Tanaka et al., 1975: 210 (Ishigaki and Miyako Islands; distribution, bionomics); Harrison, 1980: 84 (in part, Miyako and Yaeyama Guntô Islands); Miyagi & Toma, 1980: 83 –89, 91 (Iriomote Island; A L bionomics); Toma & Miyagi, 1986: 11, 28, 33, 41, 95, 96, 102 (Ishigaki, Iriomote and Miyako Islands; A* L* keys, bionomics, distribution); Toma et al., 1996a: 63 –71 (Ishigaki Island; L bionomics); Toma et al., 1996b: 167 –169 (Miyako Island; L distribution, bionomics); Tsuda et al., 1999: 601 –603 (Ishigaki Island; A bionomics); Somboon et al., 2001: 98 –101, 102 (figs 4–6, 8), 103–111 (Ishigaki Island; A, Ƥcibarial armature* E L, metaphase karyotype*, crossmating, D3 of 28S rDNA; designation as An. minimus species E); Toma et al., 2002a: 29 –40 (Ishigaki Island; L bionomics); Toma et al., 2002b: 146 –149 (Ishigaki Island; susceptibility to Plasmodium yoelii nigeriense); Higa et al., 2003: 257, 258, 260–264 (Ishigaki Island; bionomics); Sawabe et al., 2003: 772 –778 (Ishigaki Island; ITS1, ITS2 and D3 of 28S rDNA); Toma et al., 2003: 267–273 (Iriomote and Kohama Islands; L distribution, bionomics); Toma, 2006: 125 –127 (Miyako Island; A L bionomics); Nakagawa et al., 2009: 97 –99, 105, 106 (Ishigaki Island; bionomics).

Anopheles minimus minimus of Miyagi & Toma, 1978: 245 –247, 250 (Ishigaki and Iriomote Islands; A L bionomics).

Anopheles minimus species E of Somboon et al., 2000a: 136 (summary); Somboon et al., 2000b: 476 –478 (Ishigaki Island; crossmating, Ƥcibarial armature*); Harbach, 2004: 540, 545 (classification); Somboon et al., 2005a: 5 –9 (crossmating); Somboon et al., 2005b: 605 –608 (crossmating); Garros et al., 2006: 103–106, 108 (distribution); Sungvornyothin et al., 2006: 185 (A morphology); Harbach et al., 2007: 41, 45, 47, 49 (DNA sequence: ITS2 and D3 of 28S rDNA, COII mt DNA); Manguin et al., 2008: 490, 491 (taxonomy, distribution); Garros et al., 2008: print p. 2 (distribution).

Anopheles (Cellia) minimus of Tanaka et al., 1979: 31, 47–51, 554, figs 11, 12 (Miyako and Yaeyama Guntô Islands; Ƥ* 3 3G* L* P, keys, distribution, bionomics).

Diagnosis. Sequences for the ITS1, ITS2 and the D3 domain of the 28S rDNA and COII mt DNA (Somboon et al., 2001; Sawabe et al., 2003; Harbach et al., 2007: figs 2–6) distinguish An. yaeyamaensis from the other members of the Minimus Complex, i.e. An. minimus and An. harrisoni. Comparative anatomical studies indicate that the adult and pupal stages of the three species are essentially isomorphic in all life stages, however the number of branches of larval seta 7-C will distinguish most larvae of An. yaeyamaensis from those of An. minimus. The metaphase karyotype of An. yaeyamaensis is distinct from that of An. harrisoni (see below).

Description. ADULT (female)—As described and illustrated by Tanaka et al. (1979, available at http:// www.mosquitocatalog.org/files/pdfs/WR159.pdf), with following additional details. Head: Antennal length about 1.1 mm, flagellomeres 1–3 with pale scales on mesal surfaces. Proboscis length about 1.5 mm. Maxillary palpus 1.4–1.6 mm long, with 3 pale bands (in dorsal view), apical pale band about length of preapical dark band and slightly longer than preapical pale band. Cibarial armature: Cone filaments thornlike, relatively narrow and gradually tapered to pointed apex [filaments lancet-like in An. minimus and An. harrisoni (see Somboon et al., 2001: figs 1–8)]. Thorax: Integument brown, pleura with darker areas; scutum with broad median pale pruinose area confluent with scutellum of similar appearance; anterior promontory with long erect white falcate scales that transition into semi-erect pale golden piliform scales on acrostichal and dorsocentral areas that extend posteriorly to and then on lateral margin of prescutellar area to scutellum; long golden-brown to brown setae on acrostichal, dorsocentral and prescutellar areas, dark setae on fossal, antealar and supraalar areas. Wing: Length 2.1–3.4 mm; dark scaling very black on costa, subcosta and R–R1, subdued on posterior veins, pale scaling pale yellow, not white; humeral pale spot rarely absent and presector pale spot occasionally absent on one or both wings (in 1 of 87 and 5 of 87 feral females, respectively), vein R3 often (about 50%) with median pale spot and 1A pale fringe spot frequently (about 80%) present (Somboon, unpublished observations). Legs: Coxae and trochanters without scales; femora, tibiae and tarsi dark-scaled, apices of tibiae indistinctly pale, tarsomeres 1–4 with minute faint dorsoapical pale spots. Abdomen: Integument dark with uniform covering of golden setae. ADULT (male)—As described and illustrated by Tanaka et al. (1979), with following additional details. Head: Eyes more widely separated, decumbent falcate scales of interocular space slightly more numerous. Proboscis longer and more slender, approximately 1.4 length of forefemur. Wing: Generally paler and scaling reduced, fringe spots less distinct. PUPA—Habitus and chaetotaxy as described and illustrated for An. minimus by Harbach et al. (2006), setal branching compared with that of An. minimu s in Table 1, differing as follows. Trumpet: Length 0.34–0.39 mm, meatus 0.03–0.09 mm, pinna 0.27–0.33 mm. Abdomen: Length 1.97–2.36 mm. Genital lobe: Length 0.13–0.17 mm in female; 0.34– 36 mm in male. Paddle: Length 0.61–0.64 mm, width 0.41–0.47 mm, index 1.30–1.54. LARVA (fourthinstar)—Habitus and chaetotaxy as described and illustrated by Tanaka et al. (1979), setal branching compared with that of An. minimus in Table 2, with following additional details. Head: Slightly wider than long, width 0.57–0.62 mm, length 0.54–0.60 mm; integument with variable pattern of moderately to darkly pigmented areas; collar and mentum darkly pigmented. Seta 7-C with 17–26(21) branches [14–18(16) in type specimens of An. minimus (see Harbach et al., 2006) and 19/15 [left/right sides] in holotype of An. harrisoni (see Harbach et al., 2007)]. Thorax: Integument hyaline, smooth. Mesothorax with conjoined pair of median notal plates, sometimes also with pair of submedian notal plates; metathorax usually with separated pair of notal plates, apparently without submedian notal plates. Setae 1,2-P inserted on narrowly separated tubercles; support plate of pleural setal groups 9–12-P,M,T with short spine. Abdomen: Integument hyaline, smooth. Seta 0-III–VII well developed, normally branched, not noticeably larger on segments IV and V; seta 1-I–VII fully palmate with moderately pigmented leaflets, leaflets with distinct shoulders and long slender filaments (blades and shoulders narrower on segment I), blades usually with distal patch of darker pigment near shoulder. Pecten with long spines usually at each end and several interspersed among short spines. Saddle moderately to darkly pigmented, length about 0.22 mm. Seta 1-X single, simple, inserted on saddle; 4-X (ventral brush) with 9 offset pairs of setae, longest branches on anterior side of main stems. Dorsal and ventral anal papillae equal in length, shorter to slightly longer than saddle. EGG—In general as described for An. minimus by Reid (1968); length 0.39–0.55 mm (mean 0.45 mm); deck usually complete (Somboon et al., 2001).

Metaphase karyotype. The metaphase karyotype of An. yaeyamaensis (Somboon et al., 2001) is similar to that of An. minimus but very different from that of An. harrisoni (Baimai et al., 1996). Anopheles harrisoni has a prominent landmark of pericentric heterochromatin in the autosomes and the short arm of the submetacentric X chromosome. The ratio of the short to long arms of the submetacentric Y chromosome is about 1:1.5 in An. harrisoni, whereas it is 1:3–1: 4 in An. yaeyamaensis and An. minimus.

Crossing experiments. Crossmating carried out between An. yaeyamaensis and An. minimus (Somboon et al., 2001; Somboon et al., 2005b) and between An. yaeyamaensis and An. harrisoni (Somboon et al., 2005a) revealed postzygotic incompatibility. The former yielded hybrid progeny only when An. minimus females were mated with An. yaeyamaensis males, but the hybrid (F1) males were sterile or almost sterile with atrophied testes. No asynapsis was observed in the salivary gland polythene chromosomes of the hybrid larvae. The latter cross yielded hybrid (F1) progeny from reciprocal crosses, but the hybrid males were sterile with atrophied testes and accessory glands or were partially sterile with abnormal and inactive spermatozoa. The polythene chromosomes of hybrid larvae exhibited partial asynapsis or a fixed heterozygous inversion. Backcrosses revealed more severe incompatibility.

Molecular characterization. Three PCR-based assays have been developed that distinguish An. minimus and An. harrisoni of the Minimus Complex: SSCP-PCR of D3 rDNA (Sharpe et al., 1999), RFLP-PCR of ITS2 rDNA using the restriction endonuclease BsIZI (Van Bortel et al., 2000; Garros et al., 2004b), allelespecific PCR based on SCAR markers (Kengne et al., 2001) and ITS2 nucleotide variations (Phuc et al., 2003; Garros et al., 2004a). None of these methods included An. yaeyamaensis, but nucleotide variation observed in the D3 and ITS2 regions of rDNA and the COII locus of mt DNA (Harbach et al., 2007: figs 2, 3, 5) readily distinguish this species from An. minimus and An. harrisoni.

Bionomics. Anopheles yaeyamaensis (identified as An. minimus) played a major role in the transmission of falciparum malaria in the Yaeyama and Miyako Islands before the disease was eradicated from the islands between 1957 and 1962 (Kuroshima, 1960; Farid et al., 1966; Sakihara et al., 1994). The immature stages typically inhabit unpolluted streams, springs and ground pools near springs. Larvae and adults are found throughout the year, but densities decrease significantly during the cold season from December to February, during which individuals are larger and darker in colour (Miyagi & Toma, 1980; Toma et al., 1996a, b; Toma et al., 2002a; Toma et al., 2003; Somboon et al., 2001).

Anopheles yaeyamaensis is stenogamous (insemination rate about 50%) and females mate readily with a stenogamous strain of An. minimus in 30 cm cages (insemination rates of about 80%). Anopheles harrisoni is not stenogamous but females (about 20%) are able to mate with An. yaeyamaensis males (Somboon et al., 2001, 2005a).

Distribution. Anopheles yaeyamaensis has only been found on Iriomote, Ishigaki and Kohama Islands of the Yaeyama Island Group and Miyako Island of the Miyako Island Group in the Okinawa Prefecture, Japan. Ishigaki and Iriomote are the largest two islands of the Yaeyama Group and Miyako is the largest and most populous island among the Miyako Islands. The two island groups form the southern part of the volcanic Ryukyu Islands. The Yaeyamas are closer to Taiwan, about 125 km, than Miyako Island, which lies approximately 400 km east of Taipei, Taiwan.

Etymology. The specific name, yaeyamaensis, is a Latinised geographical name (yaeyama and the Latin suffix - ensis) denoting the type locality of the species in the Yaeyama Islands.

Type series. Eighty-nine specimens (13 Ƥ, 13 3, 26 Le, 26 Pe, 11 L). Holotype, Ƥ (no. 5), with LePe on microscope slide, JAPAN: Ryukyu Archipelago, Ishigaki Island, Ishigaki City, Nosoko, Nishihama stream, 6 July 2006 (Somboon et al.) (BMNH). Paratypes, same data as holotype, 12ƤLePe (nos. 2, 3, 8, 10, 11, 13, 15, 19–22, 24), 13 (no. 18), 123LePe (nos. 1, 4, 6, 7, 9, 12, 14, 16, 17, 23, 27, 32), 1LePe (no. 25) and 11L (nos. 1–11). All specimens are deposited in the Natural History Museum, London (BMNH).