Table Of ContentPROC. ENTOMOL. SOC. WASH.
108(1), 2006, pp. 198-209
ANOPHELES (CELLIA) MINIMUS THEOBALD (DIPTERA: CULICIDAE):
NEOTYPE DESIGNATION, CHARACTERIZATION, AND SYSTEMATICS
RALPH E. HARBACH, EMMA PARKIN, BIN CHEN, AND ROGER K. BUTLIN
(REH) Department of Entomology, The Natural History Museum, Cromwell Road,
London SW7 5BD, U.K. (email: [email protected]); (EP) School of Biology, Uni-
versity of Leeds, Clarendon Way, Leeds LS2 9JT, U.K. (email: [email protected]);
(BC) current address: College of Plant Protection, Southwest Agricultural University,
Chongqing 400716, PR. China (email: [email protected]); (RKB) current address: An-
imal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, U.K. (email:
r.k.b@ suheftfielldi.acn.uk )
Abstract.4A neotype for Anopheles (Cellia) minimus Theobald, the nominotypical
member of a malaria vector species complex in the Oriental Region, is designated from
a series of nine individually reared specimens collected at Ham Hang Mei, New Terri-
tories, Hong Kong, China. A hindleg taken from each of the specimens was used for
DNA extraction. The specimens were identified as species A of the complex based on
sequences for the third domain (D3) of the 28S rDNA locus and the mitochondrial cy-
tochrome oxidase subunit II locus (COID. The species is described from the neotype
series, the larval and pupal stages of the neotype are illustrated, and sequence data are
provided for the D3 and COI! loci.
Key Words: Anophelinae, D3 sequence, COI sequence, mosquito, taxonomy
Anopheles minimus Theobald is widely cies A and C based on electrophoretic data.
distributed in hilly areas throughout much Sharpe et al. (1999, 2000) confirmed the
of the Oriental Region, extending north- presence of species A and C in western
ward to about 32°30'N in China, westward Thailand and suggested the possible pres-
to Uttar Pradesh in India, southward ence of a third species (specimen #157).
through peninsular Malaysia and eastward Chen et al. (2002) concluded that forms A
to the Ryukyu Archipelago of Japan. It is and B of Yu and Li (1984) and Yu (1987)
regarded as an important vector of human in China are morphological variants of spe-
malaria throughout its distribution (Ho and cies A, and that subspecies x of Baba
Feng 1958, Reid 1968, Harrison 1980, Har- (1950) in southern China probably refers to
rison et al. 1991, Chen et al. 2002). Green An. aconitus Donitz. Recently, Somboon et
et al. (1990) showed that An. minimus con- al. (2001, 2005) provided morphological,
sists of two species (denoted as species A cytogenetic, molecular, and hybridization
and C) in western Thailand based on the evidence for the recognition of another sib-
sympatric occurrence of homozygotes of ling species of the Minimus Complex on
two enzyme loci in the absence of hetero- Ishigaki Island, Japan, which they infor-
zygotes. Green and colleagues (see Baimai mally designated as species E.
1989) also recognized a third species (de- Anopheles minimus species A appears to
noted as species D) in sympatry with spe- be the predominant species of the Minimus
VOLUME 108, NUMBER | 199
Complex in the Oriental Region. It is re- cally identified as species A from as near
corded from northeastern India to eastern as practicable to the original type locality
China and southward from Sichuan Prov- must be designated to fix the application of
ince of China through Laos, Thailand, Vi- the name. Consequently, the purpose of this
etnam, and Cambodia (Subbarao 1998; Van paper is to fix the identity of An. minimus
Bortelyer ial A1999:5 2000:4 Kengne)fethal: s.s. aS a foundation for further studies and
200iewChen <etrals12002):..Sharpe, 8etal the formal taxonomic recognition of other
(2000) estimated that the long-term effec- species of the complex.
tive population size of species C should be
approximately half that of species A. This MATERIALS AND METHODS
is concordant with its smaller range: it is
This study is based on nine adult mos-
only recorded from western and northern
quitoes, with associated larval and pupal
Thailand (Green et al. 1990, Sharpe et al.
exuviae, that were reared from fourth-instar
1999), northern Vietnam (Van Bortel et al.
larvae collected at Ham Hang Mei, New
1999, 2000; Kengne et al. 2001), south-cen-
Territories, Hong Kong, China (22°13'N,
tral provinces of China (Chen et al. 2002),
114°139E) by Leopoldo Rueda and James
and northern areas of India (Chen et al.
Pecor of the Walter Reed Army Institute of
2006). In China, species A occurs in sym-
Research, Washington, DC. Observations
patry with species C in Yunnan and Gu-
of adults were made under simulated natu-
angxi Provinces and extends eastward and
ral light. Larval and pupal chaetotaxy were
southward into Guangdong, Hainan, and
studied using differential interference con-
Taiwan Provinces.
trast microscopy. Measurements and counts
Anopheles minimus was named and de-
were taken from all specimens. Unless in-
scribed by Theobald (1901) from a single
dicated otherwise, numbers in parentheses
female collected in Pokfulam, Hong Kong,
represent modes of the reported ranges. The
but the specimen no longer exists (Theo-
morphological terminology used in the spe-
bald 1910, Yamada 1925, Harrison 1980).
cies description follows Harbach and
Harrison (1980) stated: <8If a neotype is
Knight (1980, 1982). The specimens are de-
ever needed, numerous adults with associ-
posited in the National Museum of Natural
ated immature skins collected only 20422
History (NHNH), Smithsonian Institution,
km from Pokfulam (Hong Kong Island) in
Washington, DC.
Sai Kung District, New Territories, are de-
A hindleg was removed from each of the
posited in the USNM.=9 Sharpe (1997) ex-
nine adults for DNA extraction. DNA was
amined some of those specimens but was
extracted individually from each leg using
unable to determine whether they were rep-
the procedure developed by Linton et al.
resentatives of species A or C. She attempt-
(2001) and amplified with the PCR primers
ed to sequence the D3 region of 28S rDNA
for part of the mitochondrial COII gene and
from several specimens but the sequences
the nuclear 28S ribosomal DNA D3 region
turned out to be those of a yeast-like or-
used by Chen et al. (2002). Products were
ganism rather than a mosquito. Because
sequenced on an Applied Biosystems
species A is known to have a much wider
ABI377 using the PCR primers and manu-
distribution than species C, which has not
facturer9s protocols.
been found in eastern China, it has been
suggested that the former is likely to be
TAXONOMIC TREATMENT
conspecific with the species originally de-
Anopheles (Cellia) minimus Theobald
scribed as An. minimus from Hong Kong
(Harrison et al. 1991). However, before the 1901. Anopheles minimus Theobald, 1901:
name of minimus can be unambiguously as- 186 (published 23 November). Neotype
signed to species A, a neotype unequivo- (hereby designated), female (CH643)
200 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
with associated fourth-instar larval and relationships); Phuc et al. 2003 (ITS2
pupal exuviae on microscope slide: CHI- rDNA, multiplex assay); Van Bortel et al.
NA, Hong Kong, New Territories, Ham 2003 (Odh locus, population genetics);
Hang Mei (NMNH). Garros et al. 2004 (ITS2 rDNA, single
1901. Anopheles vincenti Laveran, 1901: multiplex assay); Garros et al. 2005a
993 (published 29 November). Syntypes, (COI mtDNA, ITS2 rDNA, D3 rDNA,
5 females: VIETNAM, Tonkin, Van-Linh phylogenetic relationships); Garros et al.
(PIP). 2005b (COIL mtDNA, D3 rDNA, mor-
1902. Anopheles formosaensis I Tsuzuki, phology, phylogenetic relationships).
1902: 288. Lectotype, female: TATWAN Anopheles minimus form I of Van Bortel et
(NHM); designation by Harrison (1980). al. 1999 (Qdh locus).
1902. Anopheles christophersi Theobald,
Diagnosis.4Sequences for the D3 do-
1902: 378. Lectotype, female: INDIA,
main of the 28S rDNA locus and the mi-
[West Bengal], Duars (NHM); designa-
tochondrial cytochrome oxidase subunit I
tion by Harrison (1980).
locus (COI) for An. minimus, other mem-
1903. Anopheles aconitus var. cohaesa
bers of the Minimus Complex, and related
D6nitz, 1903: 233 (nomen novum for for-
species of the Myzomyia Series show no
mosaensis I Tsuzuki, Sep 1902, non for-
intraspecific variation, and thus represent
mosaensis II Tsuzuki, Feb 1902). Unjus-
species-diagnostic characters (see below).
tified replacement name (see Harrison
Female.4Head: Vertex with patch of
1980: 83).
pale (white to yellowish) erect scales be-
1910. Myzomyia christophersi var. alboap-
icalis Theobald, 1910: 25. Holotype, fe- hind frontal tuft, dark (brown to black)
erect scales laterally and posteriorly; inter-
male: INDIA, [West Bengal], Duars, Jal-
paiguri, Meenglas (NHM). ocular space with some white falcate scales,
several long golden-brown setae and frontal
Anopheles minimus form A of Yu and Li
1984 (morphology); Yu 1987 (morphol- tuft of long white sinuous setae. Clypeus
ogy). bare. Antenna length about |.1 mm; pedicel
Anopheles minimus form B of Yu and Li with pale integument and few minute setae
1984 (morphology); Yu 1987 (morphol- on mesal and lateral surfaces; flagellomeres
143 with pale scales on mesal surfaces. Pro-
ogy).
Anopheles minimus species A of Green et boscis about 1.5 mm, slightly longer than
al. 1990 (enzyme electrophoresis, mor- forefemur; prementum entirely dark-scaled,
phology); Baimai et al. 1996 (mitotic kar- scales appressed throughout except for few
yotype); Sucharit and Komalamisra 1997 slightly erect scales at base; labella pale.
(RAPD-PCR identification); Sharpe et al. Maxillary palpus 1.441.6 mm long, with 3
1999 (D3 rDNA, ASA and SSCP iden- pale bands (in dorsal view), apical and
tification); Sharpe et al. 2000 (COI preapical pale bands about length of preap-
mtDNA, ITS2 rDNA, D3 rDNA, phylo- ical dark band, narrow pale band at apex of
genetic relationships); Van Bortel et al. palpomere 2; palpomere 2 with semi-erect
2000 UTS2 rDNA, SCAR multiplex as- scales giving bushy appearance to proximal
say); Kengne et al. 2001 (RAPD-PCR as- portion of palpus; ventral surface of palpus
say); Somboon et al. 2001 (D3 rDNA, without scales. Thorax: Integument brown,
crossmating); Chen et al. 2002 (D3 pleura with darker transverse areas; scutum
rDNA, morphology, distribution); with broad median pale pruinose area con-
Choochote et al. 2002 (crossmating); fluent with scutellum of similar appearance;
Zhou et al. 2002 (COIL mtDNA, phylo- anterior promontory and antedorsocentral
genetic relationships); Chen et al. 2003 areas with long erect white falcate scales
(COIL mtDNA, D3 rDNA, phylogenetic that abruptly grade into decumbent golden
VOLUME 108, NUMBER 1 201
piliform scales on acrostichal and dorsocen- widely separated, decumbent falcate scales
tral areas that extend posteriorly to and then slightly more numerous. Proboscis longer
on lateral margins of prescutellar area to and more slender, about 1.9 mm, approxi-
scutellum; long golden to golden-brown se- mately 1.4 length of forefemur. Maxillary
tae on acrostichal, dorsocentral and pres- palpus largely dark-scaled, with pale scal-
cutal areas, dark setae on fossal, antealar ing as follows: narrow dorsolateral patch at
and supraalar areas. Scutellum with row of apex of palpomere 3, large dorsolateral
golden piliform scales adjacent to posterior patch closer to anterior than to posterior
row of long bronze to brown setae. Meso- margin of palpomere 4, and covering entire
postnotum and postpronotum bare. Ante- dorsolateral surface of palpomere 5. Wing:
pronotum without scales, with long dark se- Generally paler and scaling reduced, fringe
tae. Pleura with brown to golden setae on spots less distinct. Genitalia: Not dissected,
upper proepisternum (1), prespiracular area see Harrison (1980: Fig. 16).
(O42), prealar knob (245), upper (2,3) and Pupa (Figs. 1A, B).4Character and po-
lower (3) mesokatepisternum and upper sitions of setae as figured; numbers of
mesepimeron (447). Wing: Length about branches in Table |. Cephalothorax: Light-
2.743.0 mm; dark scaling black, stark on ly and more or less evenly pigmented, ap-
costa, subcosta and R4R,, subdued on pos- pendages and metanotum with darker areas.
terior veins, pale scaling pale yellow, not Seta 7-CT long, about twice length of 6-
white; costa with presector pale, sector pale CT, usually double, sometimes triple; 8-CT
and preapical pale spots, remigium and base single; 1O-CT with 143(2) branches. Trum-
of radius pale to presector dark spot, sector pet: Moderately pigmented; length 0.244
and accessory sector pale spots of R fused 0.40 mm (mean = 0.37 mm); meatus very
or separated by small dark spot; R, and R,,, short, 0.0440.10 mm (mean = 0.07 mm);
dark in middle, R, and R, pale at origin, pinna long, 0.2540.33 mm (mean = 0.29
and R, also pale at apex; R,,; with post- mm). Abdomen: Length 2.0942.53 mm
basal and preapical dark spots; M,.,, M,, (mean = 2.29 mm); lightly pigmented, an-
M,, M;,, and CuP pale at base and apex; terior margins of more anterior segments
M, with short and M,,, with long middle darker; terga and sterna minutely spiculate.
pale spot; mcu dark-scaled; CuP with mod- Seta O-III-VII long, usually with 2,3
erately long postbasal and preapical dark branches (144), inserted anterior to seta 2
spots and small dark spot at junction of on segment III, far laterad of seta 2 on seg-
meu; 1A with pale base followed by post- ments IV4VII, more or less directly anterior
basal dark and pale spots of about same to seta 5 on segments V4VII; seta 1-II4-VI
length, about distal 0.5 dark-scaled; pale with multiple thin flexible branches, 1-V,VI
fringe spots at apices of CuP, M,,,, M>, M,, usually and 1-VII always single, long, ex-
R,,;, R; and from R, to slightly anterior to ceeding length of following tergum; 6-1,I I
R, (total of 7 pale fringe spots). Halter: long, more than twice length of seta 7, 6-I
Pedicel pale, scabellum and capitellum often double (single to 6-branched), 6-II
dark, capitellum dark-scaled. Legs: Coxae single; 6-III4-V always and 6-VI,VII usually
and trochanters without scales; femora, tib- branched, number of branches generally
iae and tarsi dark-scaled, apices of tibiae progressively decrease from 6-III to 6-VII;
indistinctly pale, tarsomeres 144 with mi- seta 7-III4V short, normally branched, 7-
nute faint dorsoapical pale spots. Abdomen: VI,VII single, long, about length of follow-
Integument dark with uniform covering of ing sternum, 7-III4VI inserted on lateral
golden setae, without scales. side of fold line before posterior margin of
Male.4Similar to female except for ob- segment, 7-VII inserted on fold line at pos-
vious sexual differences; other differences terior margin of segment; setae 8,10,11-II
include the following. Head: Eyes more usually absent, on one side when present,
202 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
Fig. 1. A,B, Pupa of Anopheles minimus, modelled after exuviae of neotype female. A few missing setae
were drawn from a paraneotype. A, Left side of cephalothorax, dorsal to right. B, Dorsal (left) and ventral (right)
aspects of metathorax and abdomen. C, Map of southern Asia showing the type localities (see text for names)
of An. minimus and its synonyms (1, minimus; 2, vincenti: 3, formoasensis I; 4, christophersi; 5, alboapicalis).
CT = cephalothorax; Pa = paddle; T = trumpet; I-IX = abdominal segments I-IX; 0414 = setal numbers for
specified areas, e.g., seta 3-I. Scales in mm.
VOLUME 108, NUMBER 1 203
8-II usually double (1,2), 10-II represented
Pp l by alveolus in 3 specimens and double seta
Paddle
2(3-)5 in One specimen, 1 1-II single when present;
seta 9-I relatively long, about half length of
= 6-I, with 346(4) branches; 9-II,III small,
+ Healt at lest: Male peglike; 9-IV4VII long, curved, simple and
IX
a sharply pointed, length progressively in-
creasing from about 0.4 of segment IV to
0.6 of segment VII; 9-VIII slightly shorter
] lok = than 9-VII, about length of 4-VIII, plumose
Vill
with 9414(10) close-set branches arising
from broad flattened central stem; 10-II ab-
s sent. Genital lobe: Length 0.1640.18 mm
Vil 1 3 3 2 in female; 0.3340.37 mm in male, with nip-
4
3-5 3-5 1(24)4 a ple at apex. Paddle: Lightly pigmented
(hyaline), buttress and midrib slightly dark-
5 er, midrib distinct to near seta 2-Pa; length
VI 8ES2 a 3 a3e 7e ee Staite 0.6140.70 mm (mean = 0.65 mm), width
1-3 ie 0.4040.46 mm (mean = 0.43 mm), index
1.441.65 (mean = 1.53); marginal serra-
- tions begin 0.2840.35 from base and end
Y~S~wYewTewr~4 ae 0.4740.54 from base where they are re-
3-74( -
2(4-)6 ~ placed abruptly by short hyaline filaments;
refractile index 0.2740.33 (mean = 0.30).
= Seta 1-Pa long, sinuous, with hooked tip,
(eee about one-third length of paddle, arising
IV
ASebgdmoemnitnsa l 6(7-)8 D reo3(n7-)(9 2 ) us from shallow emargination at apex.
Larva, fourth-instar (Fig. 2).4Character
a and positions of setae as figured; numbers
VEVVyY LF of branches in Table 2. Head: Slightly wid-
I
1(2) 12-21 = er than long, width 0.5740.66 mm (mean =
_ 0.61 mm), length 0.5440.60 mm (mean =
0.57 mm); integument with mottled pattern
of moderately to darkly pigmented areas,
2
la with some intervening lightly pigmented ar-
I
4(6-)8 4(7-)8 3(4-)8 O( Q0-)2 0(1,O ) eas; collar and mentum darkly pigmented.
1(262-)3 2
Setae 2,3-C single, simple; 3-C 0.540.6
length of 2-C; 4-C single, reaching just be-
yond alveolus of 2-C, inserted in line with
I
n 5-C; 8-C usually dendritic, with 447(6)
5 =
5(5-)5 1(2-)6
branches; 13-C inserted posterolateral to
11-C. Antenna: Moderately to darkly pig-
mented; strong barb-like spicules on mesal
R(wonobfpoatsoAmsnoffhfmheruafpuenier e r opaeptnmnoo iaaadnrgbitpeelreceseemyh eh, ur pene seslts. e) s(E i surface, weaker ones on ventral surface;
3(4-)5 2( 4-)5 2(3-)6 length 0.2040.24 mm (mean = 0.21 mm).
Cephalothorax
Seta 1-A short, single, simple, length about
diameter of antenna, inserted about 0.3
T1.a ble SetNaoe 0 1 2 3 4 5 6 1 8 9 10 caonn=a=olno cltvu yen.ot fleudrs;o m base on outer dorsolateral surface; 4-
A with 447(5) branches. Thorax: Integu-
204 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
a
~
vA
4<44s fa)
SZZr oa :é = = ay S
SS44w4 44 ze <-> a
4 aS
4
Wi
\
0:5
Fig. 2. Fourth-instar larva of Anopheles minimus, reconstructed from exuviae of neotype female. A few
setae missing on the right side of the exuviae were drawn from the left side, and vice versa. A, Head, dorsal
(left) and ventral (right) aspects of left side. B, Thorax and abdominal segments I-VI, dorsal (left) and ventral
(right) aspects of left side. C, Abdominal segments VII-X, left side. D, Seta 1-IV. A = antenna; C = cranium;
M mesothorax; MATP = median accessory tergal plate; NP = notal plate; P = prothorax; S = spiracular
lobe; Sa = saddle; T = metathorax; TP = tergal plate; I-VIII,X = abdominal segments I-VIII,X; 0-15 = setal
numbers for specified areas, e.g., seta 5-C. Scales in mm.
VOLUME 108, NUMBER 1
205
ment hyaline, smooth. Mesothorax with
conjoined pair of median notal plates,
sometimes also with pair of submedian no-
tal plates; metathorax usually with separat-
ed pair of notal plates, apparently without
19-22(19)
submedian notal plates. Setae 1,2-P inserted
on narrowly separated tubercles; support
Vill1 ,2(114)
7-14(13) plate of pleural setal groups 9412-PM,T
== with short spine; 9-P,T relatively sparsely
= Nm 4 = plumose, 9-P with 9413(10) branches, 9-T
VII 1,2(125)- 351(-93 ()1 6) 3fc=oan ) SSS 4S w with 749(7) branches usually all arising
from distal half of rachis; 10-P, 9,10-M and
10-T long, single, simple; 11-P large, sig-
VI 1 1
1,2(125)- 12,12((117) ) 9-13(11) nificantly larger than 11-M,T, divided dis-
tally into 245(3) branches. Seta 4-M with
346 branches arising at base; 12-M single
1 1
2,3(2) 2,3(3) or double, branched at midlength. Seta 3-T
16-21(18)
with short thick stem bearing 11418(16)
lanceolate leaflets; 12-T shorter than 12-M,
ASbedgImoVem nitnsa l 1 4 4 aa with 345(3) branches from near base. Ab-
2,3(127)- 23 24(-2 )25 (-4 )
domen: Integument hyaline, smooth; tergal
plates of segments II4-VIII very large, about
0.640.7 width of segment, enclosing small
i 1,2(126)- 22(21)2 -5(3) ee median accessory tergal plate (not always
enclosed on segment II), all of segments I-4
VII usually but not always with distinct
II 4-7(5) submedian accessory tergal plates. Seta O-
II4VII well developed, normally branched,
largest on segments IV and V, inserted close
1
to posterolateral edge of tergal plate; 1-I-
4-7(5)4 -75(-68)( 6)
1 113--1260((1138)) 25-31(29) VII fully palmate with moderately pig-
mented leaflets, leaflets with distinct shoul-
1 ders and long slender filaments (blades and
1,2(2)
11-31-85((31426)-)3 4 -15((23495)-) 3 9 7-97) shoulders narrower on segment I), blades
usually with distal patch of darker pigment
Thorax 25-13,72((121,)82 ()31 -)6 1 3-6(3,44)( 3) 1 1 1 lnoenarg , sshionuglldee,r 3(-F[iVg . u2sDu)a;ll y3 -Id-oIuIbIl,eV ,VoIr ftariirpllye
(244), 3-VII triple; 9-I,I] inserted antero-
1
mesal to seta 7; 2-H, HI, VII branched, 2-IV4
312167---9323-4691(((3233(3441)))0 )
VI single (2-VI rarely double); seta 6-IV4
VI branched at base, triple (2 of 16 seta 6-
Head St VI 4-branched). Pecten plate with 12416
1217--114262-((112758())1 4)
spines (usually 14 or 15) with basal denti-
*obfip(sofnaRr9erid ata riannsvec)g ih.ed eu sa l
cles on dorsal side, long spines usually at
Setae sO each end with several interspersed among
short spines. Seta 1-S large, with 7-11
T2R(wonobfaflots.osAnamfmhaofoarpuhf eefneb ri roeunepa rmt vloon rdriangabaetteipeereceles hy mhe, h r -pun eeisetssln ). es sta r branches (usually with 7, 8 or 9 branches);
2-S small, with 7410(8) branches. Saddle
moderately to darkly pigmented, length
206 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
0.2040.24 mm (mean =*0.22 mm). Seta 1- achievable by means of genetic and molec-
X single, simple, inserted on saddle, much ular markers (see above). Having said this,
longer than saddle; 2-X with 19422(19) An. minimus is partially, albeit dubiously,
branches, most basal branches on dorsal distinguished from species C of the com-
side of rachis, relatively straight, with fine plex by the absence of a humeral pale spot
tapering tips; 3-X with 8-13 (often 11 or (HP) on the wings. Green et al. (1990)
12) long, thick, slightly curved, apically found that this spot was absent in 95% of
hooked branches; 4-X (ventral brush) with An. minimus females (as species A) but was
9 offset pairs of setae, longest branches on missing in only 22% of species C females
anterior side of main stems. Dorsal and from Kanchanaburi Province in western
ventral anal papillae equal in length, short, Thailand. Similarly, Sharpe (1997) recorded
shorter than saddle. the absence of a HP spot in 91% of An.
Molecular analysis 4Among the nine minimus (as species A) but only 37% of
mosquitoes available for study (five fe- species C collected at the same locality
imaless specimen mos= CH64Ik +355; 7, (Ban Phu Rat) visited by Green et al.
and 413; four males, specimen nos. CH64 (1990). Chen et al. (2002) noted the ab-
8, 49, 410, and 412), DNA from individ- sence of this spot in a comparable percent-
ual 45 consistently failed to amplify by age of An. minimus females (92.7%, as spe-
PCR, indicating poor DNA extraction. Of cies A) from southern China, but it was
the remaining eight specimens, six yielded missing in a significantly greater number of
good sequence traces for the D3 region, all species C females (84.4%). Finally, Van
of which matched An. minimus species A Bortel et al. (1999) observed an even higher
at all of the five nucleotide positions that degree of similarity between the two spe-
distinguish it from An. minimus species C cies in northern Vietnam where 99% of An.
(Sharpe et al. 2000, Chen et al. 2002, 2003). minimus (as form I) and 91.8% of species
Sequencing of the COI fragment proved C (as form II) lacked HP spots. From these
more difficult and only three sequences observations, it is obvious that the presence
were obtained, of which only one was of or absence of HP spots cannot be used to
good quality. Sequence from this individu- identify or distinguish the two species with
al, CH643, matched An. minimus A at the any degree of confidence. Van Bortel et al.
four sites with fixed differences between (1999) and Chen et al. (2002) also recorded
species A and species C in Thailand, in the the presence/absence of a presector pale
region of 294 bp sequenced (Sharpe et al. spot on the wings of males and females and
2000). These four bases all match An. min- showed that this character is even less re-
imus species A sequences obtained in China liable for distinguishing the two species.
(Chen et al. 2002, 2003). Based on the Anopheles minimus is very similar to
availability of COII and D3 sequences, three other species of the Myzomyia Series
specimen number CH643 was selected as that occur within its range of distribution in
the neotype for An. minimus (see above and the Oriental Region, i.e. An. aconitus, An.
below). The sequences obtained for this fluviatilis, and An. varuna. As pointed out
specimen are deposited in GenBank under by Harrison (1980), no morphological char-
accession numbers AMO039906 (D3) and acters are completely reliable for distin-
AM039907 (COID). guishing the adults of these species. Fur-
Systematics.4Anopheles minimus is the thermore, the adults of An. pampanai are
nominotypical member of a sibling species also often misidentified as An. minimus be-
complex comprised of three or possibly cause the distinguishing features of the
four genetic species (see introduction) that wings are not easily discerned. Consequent-
are virtually isomorphic; hence, unambig- ly, adults of An. minimus (as well as those
uous diagnosis of this species is only of other members of the Minimus Com-
VOLUME 108, NUMBER 1 207
plex) cannot be distinguished from _ the synonymy with An. minimus until the ap-
adults of these species with certainty with- plication of this name is resolved.
out associated larval and pupal exuviae. Anopheles christophersi and variety al-
Distinguishing morphological features are boapicalis were described by Theobald
provided in the identification keys of Har- (1902 and 1910, respectively) from speci-
rison (1980). However, because of the un- mens collected in localities of northeastern
certainties associated with morphological India that reside in present-day West Bengal
differentiation, the various types of molec- (localities 4 and 5, respectively, in Fig. 1C).
ular assays developed by Sharpe et al. Before Garros et al. (2005b) and Chen et
(1999), Van Bortel et al. (2000), Kengne et al. (2006) showed that An. fluviatilis species
al (2001), Phuc et al. (2003), and Garros et S was conspecific with An. minimus species
al. (2004) should be used for the unequiv- C, only An. minimus (as species A) was
known to occur in India. Species C is now
ocal identification of An. minimus (= their
known to have a wide distribution in the
An. minimus species A).
The names of four nominal species (one northern states of Madhya Pradesh, Orissa,
with an unjustified replacement name, see and Rajasthan. Although this species (as ei-
synonymy) are currently regarded as junior ther minimus C orf luviatilis S) has not been
reported from the more easterly state of
synonyms of An. minimus, but only one of
West Bengal, its possible occurrence in this
these, formosaensis I Tsuzuki, is certain to
geographic area cannot be ruled out. The
denote the same biological species. Tsuzuki
location of the type of An. fluviatilis is un-
(1902) described An. formosaensis I from
known (Knight and Stone 1977, Harrison
adult mosquitoes collected at an undis-
1980), and it probably does not exist. James
closed location on the island of Taiwan (lo-
(1902) apparently described this species
cality 3 in Fig. 1C). Evidence shows that
from several places in India, including lo-
species C of the Minimus Complex does
calities in West Bengal where type speci-
not extend into eastern areas of China, and
mens of An. christophersi and variety al-
only An. minimus occurs in Taiwan (Chen
boapicalis originated. Hence, it is possible
et al. 2002).
that one or more of the names fluviatilis,
The syntype specimens (adults) of An.
christophersi, or alboapicalis may apply to
vincenti were collected at Van Linh in the
either An. minimus or species C.
former French protectorate of Tonkin (Lav-
Fixing the identity of An. minimus by
eran 1901), which in 1946 formed the
neotype designation does not resolve the
northern part of Vietnam bordering on Chi-
taxonomic identity of species C, i.e., wheth-
na (locality 2 in Fig. 1C). Electrophoretic
er it should be denoted by an available
studies of the octanol dehydrogenase (Odh)
name or recognized as a new species. In
enzyme locus indicate that An. minimus and
contrast, there is clearly no available name
species C both occur at this locality (Nguy-
for species E of the complex from Ishigaki
en Duc Manh, personal communication).
Island, Japan, and it should be formally de-
Whether some or all of the syntypes of An. scribed and named as a species new to sci-
vincenti are conspecific with either An. min- ence:
imus Or species C is unanswerable. As in- Material examined.4Nine individually
dicated above, adults of these species are reared specimens (5 females, 4 males), each
indistinguishable in northern Vietnam, and with associated larval and pupal exuviae on
molecular methods are unlikely to be useful microscope slides. Neotype, female (CH64
in resolving the identity of the syntypes be- 3), CHINA: New Territories, Hong Kong,
cause they are mounted in balsam on a sin- Ham Hang Mei (22° 139 N, 114° 139 E), 11
gle microscope slide (Harrison 1980). For April 2002, stream margin in full sun with
these reasons, vincenti should remain in dead leaves (L. Rueda & J. Pecor). Other
