". journal of HeredIty 2007:98( I): 51-59dOl: IO.1093/)heredlesI037Advance Access publication December 7, 2006

Published by Oxford University Press 2006.

Intragenomic rDNA ITS2 Variation in theNeotropical Anopheles (Nyssorhynchus)albitarsis Complex (Diptera: Culicidae)CONG LI AND RICHARD C. WILKERSON

From the Department of Entomology, Walter Reed Army Institute of Research. 503 Robert Grant Avenue.Silver Spring. MD 20910·7500.

Address correspondence to Dr. R. C. Wilkerson. Smithsonian Institution. Museum Support Center, 4210 Silver Hill Road,Suitland, MD 20746. or e-mail: wilkersonr@Si.edu.

AbstractWe cloned and sequenced the rONA internal transcribed spacer 2 (lTS2) of 4 species belonging to the neotropical Al1ophr/u(fvyuOrJD'IUIJII1) ,tlb;ltmi/ complex, that is, A. a/bilarJir, A. a/bilar/;J B; Allopht/u lnart!ioara, a proven malaria vector; and Allopht/e!dram'0l71111, a suspected vector. Even though the ITS2 sequences of these species were vel'}' similar (~1.17% divergence),we found differences suitable for species identification and intragenomic variation of possible consequence in phylogeneticreconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existenceof partially correlated subsets of clones in A. a/bilar/is is hypothesized either to be separate rONA loci or to be semi­independently evolving portions of a single rONA locus. No differences were found between males and females, suggestingthat similar rONA arrays exist on both the X and Y chromosomes. In addition, highl~' variant clones, possibl). pseudogenes,were found in A. mara/oora from Venezuela.

Coner·rted evolution is the process where all members ofa multico~\' gene family are converted to the same sequence.The mechanism of concerted evolmion has been attributedto either unequal crossing over 01' gent: conversion (Smith1976; Zimmer et al. 1980; Dover 1982). The rONA is a mul­licopy gene famil)· t1lat exists as one or more tandem arrays ofmany transcriptional units per cell (Gerbi 1985), where con­certed evolution rapidly spreads mutations to all members ofthe gene family, even if arrays are located on different chro­mosomes (Dover 1982; Gerbi 1985; Tautz et al. 1988). Inmosquitoes, each rONA transcriptional unit is composedof an external transcribed spacer, an 18S subunit, an internaltranscribed spacer 1 (lTS1), a 5.8S subunit, an ITS2, anda 28S subunit. The rONA units within an array are linkedto each other by an intergenic spacer (lGS). The transcribedspacers are thought to contain conserved structures impor­tant in forming the mature ribosomal amplicon (Gerbi 1985;Thweatt and Lee 1990; Wessoll et al. 1992; Paskcwitz et al.1993; van Nues et al. 1995). The rONA sequence is a valuablesource of information because the functional regions thatproduce the ribosomes are highly conserved but the tran­scribed and nolltranscribed spacers have high interspecificand low intraspecific variability, making them useful iorexplaining relationships of recenliy dive"ged species and :liso

useful as a basis for polymerase chain reaction (PCR) iden­lific~oon of morphologically similar species. As such, ITSland ITS2 have been used extensively in phylogenetic recon­stmcoon of closely related and cryptic species complexes, aswell as in the de\'e!opmenr of diagnostic species-specificPCR-based markers. However, because PCR can amplifyall sequences of ITS present within the genome, variationamong ITS sequences within individuals or species could re­sult in inaccurate phylogenies and erroneous markers for spe­cies diagnostics. Consequently, identif}ing and quantifyinglevels of inrragenomic and intraspecific variation amongITS sequences arc of real importance.

The mosquito genus Anophr/a (443 formally named spe­cies) contains all the \"ectors of human malaria parasites. Be­cause many of the primary \'ectors belong to cryptic speciescomplexes, it is necessary to have accurate phylogeneticreconstructions and species diagnostics for the study ofmalaria transmission and its rclation to Allophr/u evolution.Sequences of ITS] and ITS2 are an exceUem source for suchinformation. However, in Allopb,/er, there are examples ofrONA intragenomic variation (\X'ilkerson et al. 2004; Fairleyet al. 2005), but its prevalence and magnitude is not well stud­ied. A consideration in search of an explanation for AnopheleJintragenomic rONA sequence variation is the possibility that

SI

Report Documentation Page Form ApprovedOMB No. 0704-0188

Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering andmaintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, ArlingtonVA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if itdoes not display a currently valid OMB control number.

1. REPORT DATE 2006 2. REPORT TYPE

3. DATES COVERED 00-00-2006 to 00-00-2006

4. TITLE AND SUBTITLE Intragenomic rDNA ITS2 Variation in the Neotropical Anopheles(Nyssorhynchus) albitarsis Complex (Diptera: Culicidae)

5a. CONTRACT NUMBER

5b. GRANT NUMBER

5c. PROGRAM ELEMENT NUMBER

6. AUTHOR(S) 5d. PROJECT NUMBER

5e. TASK NUMBER

5f. WORK UNIT NUMBER

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Walter Reed Army Institute of Research,Department ofEntomology,Silver Spring,MD,20910

8. PERFORMING ORGANIZATIONREPORT NUMBER

9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)

11. SPONSOR/MONITOR’S REPORT NUMBER(S)

12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release; distribution unlimited

13. SUPPLEMENTARY NOTES

14. ABSTRACT see report

15. SUBJECT TERMS

16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT Same as

Report (SAR)

18. NUMBEROF PAGES

9

19a. NAME OFRESPONSIBLE PERSON

a. REPORT unclassified

b. ABSTRACT unclassified

c. THIS PAGE unclassified

Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18

Journal of Heredity 2007:98(1)

rONA arrays are linked to different sex chromosomes, thatis, they have been found only on the X of some species(Collins et al. 1989) or on both the X and Yof others (Marchiand Pili 1994). Consequently, rONA arrays on sex chromo­somes that exhibit limited recombination could result in in­complete homogenization.

In this study, we examine ITS sequences from multipleindividuals of 4 closely related species of the neotropiealAll0phe/es a/bilanis complex. These include Anopht/es marajoaraGalvao & Damesceno (Brazil, Venezuela, Colombia, andsouthern Central America), a known carrier of malaria (Connet al. 2002); Anophdes deaneon/III Rosa-Freitas (northernArgentina to western Brazil), a suspected malaria vector(Klein, Lima, and Tada 1991; Klein, Lima, Tada, and MiUer1991); and 2 other species A. a/bilams Lynch-Arribalzaga(southern Brazil, northern Argentina, and Paraguay) andA. a/bilarsis 8 (south, central, and eastern Brazil), whose rolein malaria rransmission is unclear. The 4 species can be re­liably separated by random amplified polymorphic DNA(RAPD) (\'V'ilkerson, Gaffigan, and Lima 1995; \VJlkerson,Parsons, et aI. 1995) and white gene (Merritt et al. 2005).We initiaUy sought to examine the phylogenetic relationshipsamong these species employing a number ofgenes, includingITS2 and cytochrome oxidase I (COl) (\'V'i1kerson et aI.2005). We observed ambiguous results from direct sequenc­ing; thus, we sought to clone and sequence ITS2 sequencesfrom these species to quantify the magnitude and prevalenceof intragenomic ITS2 variation and determine its effect onphylogenetic reconstruction. In addition, we sampled bothmale and female individuals within each species to investigatepossible rONA gender differences.

Materials and MethodsTaxon Sampling

Morphological identification of A. a/bilaT'J;s s.l. was carriedout using characters found in Linthicum (1988) and Peytonet aI. (1992). Specimens used for cloning and sequencing aregiven in Table l. They represent examples from progenybroods reported in Wilkerson, Gaffigan, and Lima (1995)and Wtlkerson, Parsons, et al. (1995) from widely separatedparts of the species ranges, including type localities of the3 named species, and both sexes. In addition, a larger sampleofA. mar'!ioara is represented because of its wide distributionand the possibility of a cryptic species, A. a/bilaT'Jis E (Lehret al. 2005). For brevity, letter designations are sometimesused that follow those in Wilkerson, Gaffigan, and Lima(1995) and Wilkerson, Parsons, et al. (1995): A =A. a/bi/arR!,B = A. a/bi/am! B, C = A. martifoara, 0 = A. deallton/m, forexample; in Tables 1-3. The ITS2 sequence reported herewas used to design diagnostic primers (Li and Wilkerson2005) that correcdy identified all specimens first recognizedwith RAPD markers (Wilkerson, Gaffigan, and Lima 1995;Wilkerson, Parsons, et aI. 1995) as follow: A. a/bi/lmis,n = 56; A. marajoara, n = 407; A. dealleomm, n :: 41; andA. a/bi/tlT'Jis B, n = 56. Because there was complete concor­dance of data setS for a relatively large sample from many

52

locations, we were able to base our conclusions on a muchsmaller number of cloned individuals.

DNA Processing

DNA was isolated from individual adult mosquitoes byphenol-ehloroform extraction as described in Wilkersonet aI. (1993). The ITS2 region was amplified using PCR prim­ers based on conserved sequences in the 5.BS and 28Sribosomal subunits of A. fjlladrimamhrll/s Say (Cornel et aI.1996). The boundaries of the JTS2 were determined as inCornel et al. (1996, Figure 1A). PCRs were carried out as de­scribed in Li and Wilkerson (2005). Amplified PCR prod­ucts were cleaned using QIAquick PCR purification kit(promega, Madison, Wl). About 200 ng of each purified PCRproduct was ligated into pCR-TOPO plasmid (Invitrogen,Carlsbad, CA). Two microliters of the ligation reaction mix­ture was then transformed into competent One Shot cells(fOPO TA Cloning Kit, Invitrogen). Transformed cultureswere plated on Luria-Benani plates containing 5-bromo-4­chloro-3-indolyl-beta-D-galactopyranoside, isopropyl-beta­D-thiogaIaetopyranoside, and 50 J.lg/ml ampicillin. Successfulinsertions are confirmed by PCR. Plasmids were extracted bythe mini-prep method (Sambrook et al. 1989). Sequencingand alignment were as described in Li et aI. (2005). SequenceStatistics were obt:lined using PAUP version 4.0b4 (Swofford1998). GenBank accession numbers are given on Table 2.

Genetic Distance and Phylogenetic Analysis

Uncorrected "p" pairwise distances were calculated by PAUPversion 4.0b10 (Swofford 1998). The aligned ITS2 sequenceswere analyzed by maximum parsimony (MP) as implementedin PAUP and Bayesian analysis carried out using MRBAYES3.1 (Huelsenbeck and Ronquist 2001). The parsimony andBayesian analyses were chosen because gap informationcan be incorporated into both. Each gap was treated as a sin­gle character regardless of the length of the gap, under theassumption that a given gap is a result from one mutationalevent (Simmons and Ochoterena 2000). Single unique mura­tions were disregarded because of the possibility that theywere the result of Taq replication error. Parsimony anal}'siswas conducted using the heuristic search option with TBR(tree-bisection-reconnection) branch-swapping algorithm.Parsimony bootstrapping was done with 1000 pseudorepli­cates with 10 random taxon addition replicates per pseudor­eplicate. For Bayesian analysis, we used MJUI,IODELTEST2.2 (Nylander 2004) to choose an input evolutionary model.Markov chain Monte Carlo runs were 2 x 106 generationslong with sampling every 5 x to) generations, for a totalof 4001 samples. Of these, the first 1001 were discardedas burn-in, which is well past the point where the likelihoodplot reached a plateau.

RNA Secondary Structure

The put:ltive secondary structure of the ITS2 was esti­mated using ~WOLD (Zuker et al. 1999). A /-distribution was

Li and Wilkerson· Anopheles ITS2 Intragenomic Variation

Table I. CoUection localities, number of clones, and Gen8ank accession numbers for specimens used in cloning of rDNA 11'52 ofspecies belonging to the AnophtltJ (Nyuo,*:!n£hul) a/Mlan;J complex

Species (M = male,F = female) Code Country State Locality Coordinates

oIbilnrtiJ (A 1) BR504(S) Brazil Parana Near Guaira 24°04'5, S4°15'~a/bilami (AM2, AF2) AR7(S) Argentina Buenos Aires Baradero (type localit}') 33°4S'5, 59°30'~a/bilan;J B (B I) BR019(12) Brazil Ceani Paraipaba 3°25'5, 39°13'~oIbilanil B (8M2, BF2) BR/SP SOO(I) Brazil Sao Paulo Near Regislro 24°37'5, 47°53'~Hlarfljoara (C1) BR026(12) Brazil Amazonas Manaus 2°53'S, 60015'~marqjoara (CF2, CM2) BR;R001(10) Brazil Pani Maraje> Island (t}'pe localit}') 1°00'S, 49°3Q'Wmarajoara (C3) COJ9 Venezuela Cojedes Finca "Rosa Blanca" Not knownmarfljoara (C4) COJIO Venezuela Cojedes Finca "Rosa Blanca" Not knownmarfljoara (CS) BR4 Brazil Roraima Boa Vista 2°4S'2S"N,600 42'18"Wmarfljoara (C6) PIS9 Brazil Amapa North of Amapa Not knownmarfljoara (C7) ITB13763 Brazil Para Near ltairuba Not knowndialltomm (D1) BR;R007(lS) Brazil Rondonia Guajaci Mirim 10°50'5, 65°20'Wdiantomffl (OF2, D1\12) AR3(4) Argentina Corrientes Corrientes 27°2S'S, 59°50'Wntantorum (DF3) AR2(3) Argentina Comentes 90 km West of Posadas Not known

tener designations, A\. A2, elC, correspond 10 Table 3.

calculated to compare the minimum free energy levels of allclones given br MFOLD (Sokal and Rohlf 1981).

Results

We cloned ITS2 PCR products from each sex ofA. a/bi/aniJ(II = 3), A. a/hi/ani; B (II = 3), A. dta1leOnllH (II : 4), andA. marajoara (n =8). Individuals from widely separated local­ities, including type localities, were used as described aboveand in Table 1. The larger sample of A. 11Iarajoara served to

test for consistency ofsequence in this widely distributed spe·cies and 10 test the hypothesis of the fifth species (Lehr et a!.2005). The number of clones from the 18 total individualsranged from 3 to 28 (fable 2), giving a lotal of 217 clones.Alignment of sequences was straightforward because therewas litde sequence variation. Unless otherwise stated, the fol­lowing description does not apply to 2 variant A. marajoaraclones, C3.1 and C4.I, from individuals COJ9 and COJ 10from Cojedes, Venezuela (fables 1 and 2), which we discussseparalely.

Inter- and Intragenomic Variation

Total length of the ITS2 ranged from 344 to 365 br. Therewere 4 microsatellite regions, (G1)S-7 al posilion 118,(GAh_1I at 273, (0)4 at 147, and (Gq) at 345, all of whichwere common to all 4 species (fable 2). The first 2 regionswere variable and contributed to all the length and intrageno­mic variations of ITS2 \\;thin A. a/bi/anis Band A. dtt11ltortim.However, repeat number was not species specific. Therewere 3 interspecific and/or intraspecific 2- or 3·base indelsat positions 34, 271, and 236 and 8 single.base substilutionsat positions 30, 43, 80, 248, 260, 268, 276, and 328.

The polymorphic ACC and GC indels in A. a/bitarsis oc­curred concordandy in clones from 2 individuals in aboutequal proportions, 5 of 15 in specimen A1 and 5 of 20 inspecimen Afo.12. The third individual ofA. a/bi/aniJ (AF2) alsohad a low proportion of ACC indel clones (1 of 21), bur Ihe

GC indel was not present in our sample. There was no in­dication of any obvious correlation of the other 2 pol}'fllor­phic sites (positions 43 and 328) in this species with eacholher or the ACC and GC indels.

Phylogenetic AnalysiS

MP and Bayesian analyses were carried out for clones from3 individuals ofeach species with the microsatellite regions re­moved and indels coded as 0 or 1 (Simmons and Ochoterena2000). For c1arit}', because the combined and separate resultswere nearly identical, we present results from a single indi­vidual (Figure 1) of each species. Tree topology was the samefor both analyses, but branch support was berter with Bayes­ian analysis (suPPOrt for both shown in Figure I). Anophe/udeafleort/ol, A. 11Iarajoara, and A. a/hi/aniJ B all clustered intoseparate groups. However, A. a/bi/arli; clones separated into2 groups corresponding to the correlated and partially corre·lated ACC and GC indels described above. Variation amongclones was slight, with intragenornic base differences rangingfrom 0.0% to 0.57%, intraspecific variation ranging from0.0% to 0.60%, and interspecific variation ranging from0.28% 10 1.17% (fable 3).

Additional A marajoora Clones

Twent}'-sLx clones from 3 individuals representing A. a/bi/ani;E of Lehr et al. (2005) were sequenced, 1 from Boa Vista innorthern Brazil and 2 from Venezuela. Except for raremUlations, sequences of these clones matched sequencesfrom other collection sites, including the type locality ofA. marojoara, Marajo Island, Brazil (fable 2).

Variant A marajoara Clones

Significant divergence was seen in a single clone from 2 indi­viduals, COJ9 (clone C3.1) and COJIO (done C4.1), fromCojedes, Venezuela. These sequences were similar to eachother but quite different from all other clones (fable 2).

53

III '-• Table 2. The rDNA ITS2 sc:quc:nce~ that differ among Anophtlu alhi/anis complex species 0c:3

PositionQ.

Inidal dG GenBank .g,Specimen H- 118- 162, 236- 269- 271- 273- 326, (kcall Allele Accession :t

IIINo. Ratio 30 3S 43 57 64 80 110 118 120 129 131 171 181 207 213 238 248 250 2S8 260 268 270 2n 276 294 323 327 328 338 344 mole)" name No. ;0

Q.

AU 3/15 T .- C G T G G G G T (OT). C A G G ACe G C G C T GG GC A (GAh e /\C G G A -185.0 al AYII211321 .:;:A1.2 2/15 T - T G T G G G G T (GT), C A G G ACC G C G C T GG GC A (GA), C AC G G 1\ -182.2 112 AY828320 .....

8AI.3 9/15 T - T G T co G G G T (GT), C A G G - G e G C T GG .. A (GA), C Ae A G II -185.5 a) AY828322 -...IAI.4 I/IS T - T G T G G G G T (GT). e A G G - G C G e T GO .- II (GA), e AC A G A -185.5 a4 AY828323 ;CAM2.1 4/20 T .- C; G T G G G G T (GT). e 1\ G G ACC G e G e T GG GC A (G/\h C Ae G G A -185.0 al AY82l132I ~

AM22 1/20 T - C G T G G G G T (GT). C A G G ACC G C G C T GG GC A (GA). C Ae A G A -1111.5 as AY828335 ~

1\:'.12.] 15/20 T - T G T G G G G T (GT), C A G G - G C G C T GG .- A (GA), C AC A G /\ -18S.5 a6 AY8283361\1'2.1 20/21 T - T G T G G G G T (GT), C A G G - G e G e T GG - 1\ (GAh C lie A G A -185.5 a) AY828322AI'2.2 1/21 T - C G T G G G G T (t....1'). C A G G Ace G C G C; T GG - A (GA). C AC G A A -181.5 a7 DQll77807111.1 7/10 T - C G T G G G G T (GT). C A G G ACC A C G A T GG .. A (G/\h C I\C A G A -179.7 /1/ AY82832481.2 1/10 T .- C G T G G G G T (GT)s C A G G ACC A C G A T GG .. A (G/\), C I\C A G A -176.7 h2 AY82832581.3 1/10 T - C G T G G G G T (GT). C II G G ACe A C G A T CiG .. A (GA). C lie II G A -180.0 bJ IIY828326ilIA 1/10 T - C G T G G G G T (G1'). C A G G ACC A C G II T GG - A (GA), C IIC A G A -17').] b-I AY828]2781'2.1 9/10 T .- C G T G G G G T (OT). C A G G Ace A C G II T GO - A (GA), C Ae II 0 A -179.7 PI IIY8283248F2.2 1/10 T - e G T G G G G T «(""1"),, C 1\ G G ACC A e G /\ T GG .. A (GA). e Ae A G A -180.0 ItJ AY8283261l~12.1 17/18 T - C G T G G G G T (GT). C A G G ACC A e G 1\ T GG .. A (GA), C Ae A G A -179.7 /" ,\V82832·\B~I2.2 1/18 T .- C G T G G G G T (GT). C A G G ACC A C G A T GG - 1\ (GA). C Ae A G A -180.0 ItJ AY828326CI.I H/IS T CC C G T A G G G T (GT). C A G G - G C G e T GG .- G (GA). e AC G G A -178.3 ,f AY828328CI.2 1/15 T CC C G T " G G G T (GT). e 1\ G G - G e G C A GG - 1\ (GA), c: AC G G A -17(,.5 ,2 AY828329CI'2.1 2/') T ce C G T A G G G T (GT), C A G G - G C G C A GG .. A (GA), C Ae G G A -178.7 rJ AY82833?CF2.2 7/9 T CC C G T A G G G T (GT). C A G G - G c: G e T GG - G «;A). C AC G G A -178.' tI AY828328<:'\12.1 21/28 T CC C G T 1\ G G G T (GT). C 1\ G G - G C G C T G(; - G (GA). C AC G G A -178.3 (, AY828328<:'\12.2 1/28 T ce C G T ,\ G G G T (GT)s C A G G - G e G e T GG .. G (G....). c AC G G 1\ -175.6 ..., OQOn808e~l2.3 6/28 T CC e G T A G G G T (GT), C " G G - G e G e A GG .. A (Gil), C Ae G G A -178.7 rJ AY82833?0.1 1/8 T ce C A A A A T C (GT), T e C /I - G T T C T - - /I (Gil), T IT A T G -164.3 ·(f AV828344C3.2 7/8 T CC C G T A G G G T (GT). C A G G - G C G e T GG - A (GA)" C AC G G A -1n,2 as AY82834SC4.1 1/4 T CC C G T " G G G T «,"T)s C A C /I - G T T e T .. .- A (G/\), T IT A T G -169.5 (7 IIY828346C4.2 3/4 T CC C G T A G G G T (GT),. C A G G - G C G e T GG - A (GA). C AC G G A -177.2 (8 ,\\,828.147C5.1 2/4 T CC c: G T " G G G T (GT), C 1\ G G - G C G C T GG .. A (GA). e Ae G G A -179.0 t9 AY828348C5.2 1/4 T CC C G T A G G G T (GT). C A G G - G C G C T GG .. ,\ (GA)" C AC G G A -1n,2 as AY828345C5.3 1/4 T C:C c: G T " G G G T (GT), C A G (; - G e G c: A GG - 1\ (GA), C AC G G A -178.7 rJ A\'828339C6.1 1/7 T CC C G T 1\ G G G T (GT), C " G G - G C G e T GG .- A (GA). C AC G G A -179.0 t9 ,/\Y828348C6.2 1/7 T CC C G T A G G G T «(,,'T), C A G G - G C G C A GG - A (GI\), C AC G G A -\78.7 rJ ,W828339C6.3 5/7 T c:C e G T A G G ti T (GT). e A G G - G C G C T GG .- A (GA). e AC G G A -1n,2 as AYS28345C7.1 3/3 T ce C G T A G G G T (GT), C A G G - G C G e A GG - A (GA), e AC G G A -178.7 ,J AY82833?01.1 4/11 e CC C G T G G G G T (GT). C A G G Ace G c G C T CrG .- A (GA). e Ae G G A -182.4 til IIY82833001.2 1/11 C ec e G T G G G G T (GT). C 1\ G G Ace G c G C T GG - A (GA), e Ae G G A -180.3 J2 AY82833I1)1.] 5/11 C CC C G T G G G G T (GT). C A G G Ace G e G C T GG - /\ (GAl, C IIC G G " -178.6 tlJ AY82833201.4 1/11 C CC C G T G G G G T (GT)" C A G G Ace G e G e T GG - A (G,\)" e I\C G G ,\ -183.4 JI AY828333DI'2.1 10/14 C CC C G T G G G G T (GT). C A G G Ace G C G e T GG - A (GI\). e AC G G A -182.4 JI AY82833001'2.2 4/14 e cc C G T G G G G T «,"1),. C A (i G Ace G C G e T GG - A (GA), e AC G G A -178.6 tlJ AY828332-, DM2.1 6/12 C CC c: G T G G G G T (GT)" C A G G I\CC G c: G C T GG - A (GA). e Ae G G A -1112.4 til AYII283300~12.2 6/12 C ce e G T G G G G T (GT),. C A G G Ace G C G C T GG .- A (GA), C AC G G A -178.6 tI) AY828332DF3.1 1/8 C CC c: G T G G G G T (GT)/ e A G G ACC G C G e T GG - A (GA). C AC G G A -182.4 til I\Y82833001'3.2 3/8 c: CC C G T G G G G T (GT). e ,\ G G Ace G e G C T GG - A (GAl> C Ae G G A -\78.6 JJ AY828332DF3.3 4/8 C CC C G T co G (i G T (GT),. c: A G G Ace G C G C T GG .- A (GA), C AC G G A -180.3 J2 AY828]31

Spccil'S ...... fon",," A =,1. 6IbiJlU7;'. Il = ,1. 6/bi/QrJiI Il, C = ,t••phd" I• .,.,¥OII"d. D =A.op"'''' "46"""11. C"lumM I an,1 2 a", ...,..,imen number, clon. numb.... and number of clon"/'OIa1, for exampl., AU i. A. • /bilanu 'p«:im.n J•••1oflike clon•• number I, ",hich was fm",,1 in 3"f 15 ,,>tal clon••. ~I and I'd..,,,,. mol. and fmtal•• ir!<nown. Onl)' ,·..iabl."'•••••'" .he,,,,,,,. Single mUI.tion. Ihal a!'P""'a1 only onc. "'('f<: nOI consid....d boc.u", of Ih. ",,"ibili,y .he)· """" due 10 7;'"M'fnrs•

• Th. initial ,IG is d,. energy 1<\'<1 or folded RN,\. Th. ",gion ror Ih. Ie.. includes 91 "'..... in lh. S.BS .ubunil and 43 b•••• in .h. 285.

Li and Wilkerson· Anopheles ITS2 Intragenomic Variation

Table 3. Uncorrected "1''' distlnce matrix of clones from AI, BI, CI, and 01

AI.! A1.2 AU AlA 81.1 81.2 81.3 81.4 CI.I CI.2 01.1 01.2 01.3 01.4

A1.2 0.0028AU 0.0057 0.0028AI.4 0.0057 0.0028 0BU 0.0085 0.0113 0.0086 0.0086B1.2 0.0086 0.0114 0.0087 0.0086 0BU 0.0057 0.0085 0.0057 0.0057 0.0028 0.0029BIA 0.0086 0.0114 0.0087 0.0086 0 0 0.0029CI.I 0.0057 0.0086 0.0115 0.0115 0.0143 0.0144 0.0115 0.0144Ct.2 0.0028 0.0057 0.0086 0.0085 0.0114 0.0114 0.0086 0.0115 0.002901.1 0.0028 0.0057 0.0087 0.0087 0.0114 0.0115 0.0086 0.0114 0.0085 0.005701.2 0.0028 0.0057 0.0088 0.0087 0.0116 0.0117 0.0087 0.0116 0.0087 0.0058 001.3 0.0028 0.0057 0.0088 0.0088 0.0117 0.0118 0.0088 0.0116 0.0087 0.0058 0 001.4 0.0028 0.0056 0.0086 0.0086 0.0113 0.0114 0.0084 0.0114 0.0086 0.0057 0 0 0

Clone C3.1 differed from other conspecific clones by 5.6- (Figure 2Q. Note that the stem and loop near the presump-6.6% and C4.1 differed b}' 3.5-4.1%. Genetic difference tive lTS2 excision sire (Fritz et al. 1994) (next to the rightbetween the 2 variant clones was 2.0'Vo. arrow in Figure 21\) is missing in the 2 variant clones

(Figure 2B,q.

Secondary Structure of rRNA

The secondary structures of rONA ITS2 were predictedby MFOLD (Zuker ct al. 1999). l\linimum free energies inkilocalories/mole were -181.5 to -185.5 for A. a/hi/arris,-176.7 to -180.0 for A. a/hi/arris B, -175.6 to -179.0for A. mar'!ioara, and -178.6 to -183.4 for A. deaneo11lm.The structures of the 2 variant A. mar'!ioara clones, C3.1and C4.1, have significantly lower energy (-167.4 and- 168.9 kcal/mole; P < O.ot in the Student's I-tesr) and pre·sumably lower stability than other A. mar'!ioara. Figure 2/\shows the predicted folding structures of all clones in thea/bilarsis complex except 0.1 (Figure 2B) and C4.1

Figure I. MP rree generated from rONA lTS2 sequencederived from individuals Al, Bl, Cl, and 01 in Table 3.Species (number of clones): Aflopbeks a/bitarsis (15), A. a/bitarsisB (10), Anopheks lI,arnjoara (15), and AHopbe/ts deantonfl!l (11).The same topology is found when AM2, AF2, BM2, BF2,CM2, CF2, OM2, and DF2 are combined. Bootsrrap values areon rhe branches. The first numbers are from MP analysis; rhesecond number in parenthesis is from I\IRBAYES analysis.

DiscussionA basic assumption about multigene families, such as rONA,is that the processes collectively referred to as concerted evo­lution (gene conversion and unequal crossing over) maintainhomogeneity of all copies (Hood et al. 1975; Smith 1976;Zimmer et al. 1980; Dover 1982). Murations rapidly spreadto all members of the gene family even if there are arrayslocated on different chromosomes (Dover 1982; Amheim1983; Gerbi 1985; Tautz et al. 1988). In the case of noncodingregions, such as ITS2, this can lead to fixed interspecific dif·ferences and intraspecific homogeneity. The efficiency ofhomogenization of rONA is usually high (Liao 1999), asexemplified by its common use as a marker for mosquitoidentification, most of which are derived from ITS2 (exam­ples given in Wilkerson et al. 2004). However, as our resultsshow, when mutation rates arc higher than rates of homog­enization, then variation within individuals may be greaterthan that observed between populations (see also Fritzet al. 1994; Onyabe and Conn 1999; Wilkerson er al. 2004).This possibilit}' should be accounted for before rONA isused for phylogenetic or population srudies or as a basis forspecies-specific PCR primers.

ITS2 Variation

The ITS2 of all 4 RAPD-determined species in rhe AlbitarsisComplex were intragenomicaUy and interspecifically variable.Length variation was limited (344-365 bp) and mostly attrib­utable to the 2 variable microsatellite regions. In addirion, .there were a number ofindels and base substitutions account­ing for both the length and sequence variabilities (see Resulrsand Table 2). Anopheles a/bi/arris differed from the other spe­cies in ha\'ing intragenomically variable ACC and GC indels(positions 236 and 271) and a variable TIC mutation atposition 43. The ACC indel and the T/e mutations were

55

· journal of Heredity 2007:9B(I)

Figure 2. Predicted secondary structure of rONA [TS2, including a combined 134 bases from the flanking 5.8S (91) and 28S (43)subunits. The secondary structure common 10 clones from all species (A) except for the 2 clones shown in (B) and (C). whichwere found in 2 individuals of Anoplx/ts marajoarn from Cojedes, Venezuda.

used b)' Li and Wilkerson (200S) to design species-specificprimers to identify A. a/bitarsis, A. albi/arsis Band A. dtantonlolas a group, and A. a/hi/ams, respectively. Even though theabove 3 (ACC, GC, and TIC) differences are not fixed inA. a/bitarsis, PCR primers designed based on them still am­plified as if there were only target sequence present and there­fore still functioned to diagnose the species or groups ofspecies.

Clones of A. a/hi/anis ITS2 showed ~7fealer diversity thanthe other 3 species. In this case, inttagenomic [TS2 variationwithin A. albi/arsis was greater than that between species inthe complex. For example, the genetic distance between A1.1and A1.3 (A. a/bi/ams) was 0.57%, whereas the differencebetween AU and 01.1 (A. dtaneomo/) was 0.28% (fable 3).This is an apparent example of mutation rates that are higherthan homogenization rates. Inttagenomic variation at ITS2,and in other parts of the rONA gene array, is probably verycommon (Harris and CrandaU 2000). In Anopheles mosqui­toes, inttagenomic variation has also been found in a num­ber of other Anopheles species (Onyabe and Conn 1999;Wilkerson et al. 2004; Fairley et aI. 200S) and in other mos­quitoes in subfamily Culicinae (Black et a!. 1989; Wessonet a!. 1992; Miller et al. 1996; Beebe et a!. 2000).

Effect of Microsatellites on Phylogenetic Reconstruction

Highly variable microsatelJites may have confounding effectson phylogenetic and population genetics anal)'ses. Harris andCrandall (2000) noted that if the multicopy nature ofa markeris not recognized, inconsistent results can occur because

S6

aUeles will not be distributed in a Mendelian manner. Cloningresults verified our hypothesis that microsatellite variationwas responsible for ambiguous sequencing results. However,in our case (data not shown), and in that of Vogler andOeSaUe (1994), ph)'logenetic results were not affected byexclusion of microsatellite regions.

Chromosome Location of rONA Arrays

Figure 1 shows 2 clusters of clones from the same individualof A. a/bi/arsis that are as different from each other as theyare from the other 3 species. This suggests either that thereare 2 rONA loci within A. a/hi/am. or that there are semi­independently evolving homologous rONA loci. This couldbe caused by inefficient gene conversion and gene recombi­nation. Multiple rONA locations are not unusual, for exam­ple, there are 5 in humans (Gonzalez and Sylvester 2001) andat least 2 in Drosophila I!Jtki (Hennig et aI. 1975) and grass­hoppers (\X'hite et al. 1982). Similar explanations were con­sidered for other A1Iophe/es mosquitoes by Onyabe and Conn(1999) and Beebe et a!. (2000).

The rONA arrays are usually on chromosomes associatedwith sex determination. Kumar and Rai (1990) and Marchiand Pili (1994) mapped dozens of species of mosquitoesand found rONA loci on the aUlOsomes of culicine mosqui­toes and on the X and Y chromosomes of an Allopht/tJ. Inaddition, they found loci on heterologous chromosomes ingenus Aedes, the only confirmed example of loci on differentchromosomes found so far in mosquitoes. In the GambiaeComplex (subgenus ullin), Allophe/es l,aolbiae Giles and

Atlopheles arabimsis Patton have rDNA only on the X chro­mosome, whereas in the other species of the complex, itis on the X and Y chromosomes (Collins et aI. 1989). In2 Anopheles subgenus N)'SJor!?Ynfhlls species, Rafael et al.(2003) found rDNA on both the X and Y chromosomes.If rDNA was associated only with the X chromosome, asit is in A. gmnbiae, then males would be expected to have halfthe number of rDNA cimon copies (Collins et aI. 1989) andhalf the haplotype diversity. If there were a subset of rONAassociated with the Y but not the X chromosome, then onlymales would be expected to have the V-associated rONA. Inour sample, we did not see higher haplotype diversity asso­ciated with males or females.

Polanco et al. (2000) proposed 2 models to account forapparently correlated sets of rONA other than loci on sep­arate chromosomes: a haplotypic single-lineage model forITS evolution and a multilineage model for IGS evolution.The X and Y chromosomes in Anopheles are only partially ho­mologous, and X chromosome variants do occur (Baimaier at. 1993; Rafael et at. 2003). Such factors may contributeto incomplete homogenization and could explain our findingof partially correlated intragenomic ITS2 haplotypes. Asemployed in the above studies, physical mapping using in sin.hybridization is needed to confirm the location of rDNA lociin the A. albilarsis complex species.

Anopheles albitlJrsis Species E

Based on complete sequence of the mitochondrial COl, Lehret al. (2005) proposed a fifth species (A. albilams E) for thealbilarsis complex in northern Brazil and Venezuela. Wefound no evidence from ITS2 sequence to support their con­clusions. Isosequential ITS2 can occur in closely relatedAnopheles species (see above), and additional data are neces­sary to resolve this question.

Variant A marajoara Clones

Anoplules nlar'!i0ara individuals COJ9 and COJI0 fromCojedes, Venezuela, each had a different highly divergentclone (fable 2). The sequences are similar to the otherA. mar'!ioara ITS2 but differ from each other by about asmuch as A. mar'!ioara does from the other 3 specie::s. One::of the clones (C3.1) has many mutations throughout itslength, whereas the other (C4.1) is the same as all the otherA. nlar'!ioara clones up until position 207, after which itmirrors the mutations in the more divergent clone. This''half-variant'' could be due to template jumping, whichcould anomalously combine normal and variant sequence::(Thompson et al. 2002). The relatively high sequence varia­tion between these 2 clones suggest.~ that these copies couldbe from nonfunctioning rONA (pseudogenes). To test thispossibility, we compared estimated minimum free energy lev­els and looked at the secondary structure predicted b)' theprogram MFOLD (Zuker et al. 1999). We found that thefolding Structures of these 2 clones have statistically signifi­cantly lower energies than all other clones (see above) andtherefore lower strUctural stability. In addition, the variamclones lack a stem and loop at the ITS2 excision site present

Li and Wilkerson· Anopheles ITS2 Intragenomlc Vanation

in all other clones (Fibrure 2). It is possible that this Structuralvariation could affect cleavage efftciency of the precursorRNA, and it leads us to conclude that these copies probablycome from nonfunctioning rDNA (pseudogenes). To ourknowledge, this is the first of such report in a mosquito, butthey have been documented in other organisms (Brownellet aI. 1983; Benevolenskaya et al. 1997; Razafimandimbisonet aI. 2004). Further work is clearly needed to verify thisobservation.

Application of ITS2 Intragenomic Variation

Unambiguous identification of Anophelu malaria vector spe­cies is essential for the study of an array of factors that affectcontrol and disease transmission. When morphological char­acters are not available, molecular alternatives must be found.In the case of the Albitarsis Complex, we initiall)' looked atsequence of the rONA ITS2 hoping to find a way to separatethe 4 species. Ordinarily, it is possible to directly sequence thelTS2 without ambiguity, but in the A1bitarsis Complex, directsequence results were not clear because of intragenomic var­iation. Using ITS2 clones, we were able to identify primerlocations that were not compromised by intraspecific andintragenomic variability (Li and Wilkerson 2005). Such var­iability often cannot be seen in direct sequencing and couldlead to design of primers that will give erroneous or ambig­uous results. For example, at position 236 (fable 2) in./1. albilarsis, there are 2 alleles, ACC present and ACC absem.In a consensus sequence, ACC absent copies are preferen­tially amplified because they arc more common. If a primerwere designed based on ACC present, then an A. albilarsissample would be misidentified as A. albilarsis B or A.dtaneoTJInl. Similar results could occur with primers designedbased on positions 43 and 328. With these data, we were ableto design primers for the 4 species previously determinedusing RAPDs and provide an identification tool for an im­portalll malaria vector group.

Acknowledgments-We greatl)· appreciate me support of those who helped collect and rear manyof the specimens used in this study. These include Terry Klein and Eric Mil­SIre)' who Were then members of the US Army Medical Research Unit in Riode Janeiro. BUI we especially thank Jose Bento Uma without whose eXiraefforts many of samples would not have been a\'ailable to us. We also thankLee Weigt at Ihe Smithsonian InstilUtion, uboratory of Analytical Biol"l:)',for use of laOOralol)' and sC<Juencing facilities; David Erickson, DesmondFoley, :md Jan Conn for thoughtful review of a draft of the manuscripl;and Carl Schlichting for advice on statistical analysis, This research ...·as per­formed under a Memorandum of Understanding between the Walter ReedArm)' Institute of Research and the Smithsonian lnstilUtion. with institu·lional support provided by both organizations, The malerial publishedreflecls Ihe ..iews of the authors and should not be coostrUed 10 representthnse of the Dcpanment of the Ann>' or the Department of Defense. This...·ork was sponsoreu by NIH AI R0154139 to Jan Conn.

ReferencesArnheim N. 1983. Concerted "'olution of nlultigene families. In: Nei1\1, Koehn RK, editors. Evolution of KCnes and proteins. Sunderland(?>.Ii\): Sinauer. p. 38-6t.

57

journal of Heredity 2007:98(1)

B2imai V, Kijchalao U, Rallarurithikul R. 1993. Metaphase karyol}-pes ofA"opbtl,J of Thailand and southeast Asia: II. Maculatus Group, NcocclJia

S<:ries, Subgenus Cti/ia. Mosq Srst. 25:116-123.

Beebe NW, Cooper RD, Foley DH. Ellis JT. 200n. Populations ofthe south-west Pacific ma!:l.ria vector .A.6phtkJ jarallh J.J. re"ealedby ribosomal DNA transcribed spacer polymorphisms. Heredily. 84:

244-253.

Benevolenskaya EV, Kog.m G[.. Tulin AV, Philipp D, Gvozde\' VA. 1997.S<:gmenled gene convenion as a mechanism of correction of 185 rRNApseudogene located outside of rDNA dusler in D. mtlan6S1'1Ur. J :-'101 E,'o!.

44:646-65 J.

mack WC 4th.I\[c1.ain OK, Rai KS. 1989. Panems of variation in the rRNAcimon within and among world populations of a mosquito A,MJ alb6PVfllJ(S1cusc). Genetics. 121:539-550.

BrOllmell E, Kt)'stal M, Amhcim N. 1983. Structure and evolution of humanand African Ape rDNA pseudogenes. Mol Bioi Evol. 1:29-37.

Collins FH, paskeuilZ SM, Finncrrr V. 1989. Ribosomal RNA genes of the

,fn""klts gambiat species complex. Adv Dis Vector Res. 6:1-28.

ConnJE, Wilkerson Rc, Segura />INO. de·Souza-Raimundo 11.. SchlichtinllCD, Winz RA, Povoa MM. 2002- Emergence of a ncu' ncotropical maJariavector facilitated br human miwation and changes in land use. Am J TropMed Hyg. 66:18-22.

Comel AJ. Poner CH, CoOins FH. 1996. Pol)1nerase chain rC'olction speciesdiagnostic assar for A"6pht1tS 'luaJril11t1Q1latllJ crn"i" species (Diptera:Culicidae) based on ribosomal DNA 1TS2 sequences. J :-.led Entomo!'

33:109-116.

Dover GA. 1982. Molecular dri"e: a cnhesive mode of species evolution.Nature. 299:111-117.

Fairley TI., Kilpatrick CWo Conn JE. 2005. Intragenomic helerogencily ofinternal transcribed spacer rDNA in neotrnpical malaria vector, An6pht/uIlI/lIaJdlis (Diptera: Culicidae). J Med Entomol. 42:795-800.

Fritz GN, Conn J. Cockburn A, Seawright J. 1994. Sequence anal)"Sis of Iheribosomal DNA internal transcribed spacer 2 from populations ofAnopht/tJ"lInttJ0ra,; (Diplen: Culicidae). Mol Bioi Evol. 11:406-416.

Gerbi SA. 1985. Evolution of ribosomal DNA. In: Madnl}'re RJ. editor.Molecular evolutionary genetics. New York: Plenum Press.

Gonzakz II.. Sy/veslerJE. 2001. Human rDNA: e"olutionat}, pallerns withinthe genes and tandem am)'S derived from multiple chromosomes. Geno·mics. 73:255--263.

Harris OJ. Crandall KA. 2000. Intragenomic variation 'Iloithin ITS I and 1TS2of fresh water crarfishes (Decapoda: Cambaridae): implications for phyloJ;e·netic and microsate:Uite studies. Mol BioI Em\. 17~291.

Hennig W. Link B, Lconcini O. 1975. The location of nucleolus otg.lnizerregions in Drotopbila Jrytki. Chromosoma. 51:57-63.

Hood I~ Campbell JH, Elgin Sc. 1975. The organization, expression, andevolution of antibody genes 2nd other multigene families. Ann Rev Genet.

9:305--353.

Huelsenbeck JP, Ronquist F. 2001. :-'IRBAYES: Bayesian inference of ph)'­

logen)'. Bioinformatics. 17:754-755.

Klein TA. LimaJBP. Tada MS. 1991. Comparative susceptibility of anoph­eline mosquitoes to plasmodium falciparwn in Rondonia BrniI. Am J TropMed Hyg. 44:59S-603.

Klein TA.l.imaJBP, Tad. MS. Miller R. 1991. Comparati"e susceptibility ofanopheline mosquitoes in Rondonia Br:azil to infection b)' plasmndium "i,·a,..Am J Trop Med H}·g. 45:463-470.

Kumar A, Rai KS. 1990. Chromosomal locali?.ation and cop)' number of18S + 285 ribosomal RNA gt:ncs in evolutionarily diverse mosquitoes(Diptera, Culicidae). Hereditas. 113:277-289.

!"ehr MA. Kilpatrick CW, Wilkenun RC, Conn JE. 2005. Cryptic specIes inthe rlnophtkl (I\!JJfDr/!}lI(blls) a/bihzrsis (Diptera: Culicidae) complex: incongru.

58

ence between random amplified pol)1norphic DNA·po1rmerase chain fe-.lC·

tion identification and anal)'Sis of mitochondrial DNA COl gt:ne sequences.Ann Entomol Soc Am. 98:894-903.

I.i C. Lee JS, Groebner JI.. Kim H, Klein T, O'Guinn MI.. Wilkenon RC.2005. A newly recogni7.ed species in the AnophkJ H)'fC:lnuS Group and mo·leeular identification of rdated species from the Republic of South KorC'ol(DiplCfa: Culicidae). Zootaxa. 939:1-8.

l..i C, Wilkenon RC. 2005. Identification ofAl1opb,kJ (NyJJori?JnthuI) alhilan;Jcomplex species (Diptera: Culicidae) using rDNA ITS2-bascd pCR primers.~'1em Inst OS"--aldo Cruz. 100:495--500.

l.iao A. 1999. MolccuIar evolution '99 Conccned evolution: molecular mech·anism and biological implications. Am J Hum Genet. 64:24-30.

linthicum KJ. 1988. A Revision of the Argrritarsis Section of the SubgenusJ'f!mr/!}lKbNs of A116J>ht1t1 Diptera Culicidae. :-'Iosq S)·st. 20:98-271.

Marchi A, Pili E. 1994. Ribosomal RNA genes in mosquitoes: localization bylIuorescence in sifll hybridization (FISH). Heredity. 72:599-605.

Merritt TJS, Young CR, Vogt RG, Wilkerson RC. Quattro JM. 2005. Intronretention identifies a malaria ,"ector within the A"ophtlts (J'f!JIDri?!lIthIlS)tJlbilimil complex (Diptera: Culicidae). 1II01 Ph}'1 Evol. 35:719-724.

Miller BR. Crabtree MB, Savage HM. 1996. Phylogeny of founeenCillo: mosquitO species, including the CIJ!tx pipimJ complex, infCrTcdfrom the internal transcribed spacers of ribosomal DNA. Insect Mol BioI.5:93-107.

Nl'!anderJA. 2004. MrModeltest ,'2. Uppsala,Swcden: E"olutionary BiologyCen,",. Uppsala University.

On)-abc DY, Conn JE. 1999. Inuagcnomic heterogenei!>, of a ribosomalDNA spacer (ITS2) varies regionally in the ncotropical malaria vectorAnophtkJ nll~"",," (Diptera: Culicidae). Insect ~lo1 BioI. 8:435--442-

pask.".'i12 SM, Wesson OM, Collins FH. 1993. The inlemal transcribedspacers of ribosomal DNA in five members of the Anopbtltl,1,lVIIbiat speciescomplex. Insect Mol BioI. 2:247-257.

PC)10n E1.. Wilkenoo Rc, Harbach RE, 1992. Comparative: analysis of the

subgenera KmtJ~ and N"ss6r/!}nrbuJ (Diptero: Culicidae). Mosq S)'St.24:51-69.

Polanco C, Gonzalez AI, Dover GA. 2000. Patterns ofvariation io the iOler·genic spacers of ribosomal DNA in DroJoph;/a mtl""of,rJlf" suppon a modelfor genetic exchanges during X·)' pairing. Genetics. 155:1221-1229.

Rafael MS, Tadci WI'. Recco-Pimcntcl SM. 2003. Location of ribosomal

gt:ncs in the chromosomes of AMphtltJ tlar/i"gi and Al10phtkJ """t1{JO~4ri

(Diptera, Culicidae) from the: Brazilian Amazon. Mem Inst OswaldoCruz. 98:629-635.

Razafunandimbison SG. Kellogg EA. Bremer B. 2004. ReceOl origin andphylogenetic utiliI}' ofdivergent ITS putative pscud"llenes: a case sNd)' fromNauclccae (Rubiaceac:). SySt BioI. 53:177-192-

Sambrook J, Fritsch EF. Maniatis T. 1989. Molecular cloning. A laboratoty

manual. 2nd ed. Cold Spring Harbor, NY: (".old Spring Harbor Laboratory.

Simmons MP, Ochoterena H. 2000. Gaps as charnelen in sequence-basedphylogenetic anal)·ses. S)'5t Bio!. 49:369-381.

Smith GP. 1976. Evolution of repeated DNA sequences br unequal cross·over. Science. 191:428-535.

Sokal RR, Rohlf 1-). 1981. Biomctry. 2nd cd. New York: \'(!.H. Freeman &

Company.

Swofford 01_ 1998. PAUP-. Phyl"llenetic anal~'Sis using parsimony (-andOIher methods), Version 4. Sunderland (1\IA): Sinauer Associates.

Taulz D. Hancock JM, Webb DA, TaulZ C. O<"'er GA. 1988. C.ompletesequences of the ribosomal fu'lA genes of DroJophila 111","6gll1f", Mol Bioi1"'01. 5:366-376.

Thompson JR, .\larcelino LA, Polz MF. 2002. Heteroduplexes in mixed·lemplale amplification. consequence and elimination br 'reconditioningI'CR'. Nucleic Acids Res. 30:2083-2088.

Th"'can R, Lee: Jc. 1990. Yeast precursor ribosomal RJ."lA molecularcloning and probing the higher onler structure of Ihe internal transcribedspacer I by kethoxal and dimeth)'lsulf3te modification. J Mol BioI. 211:305-320.

van Nues RW. RientjesJl\I. Morre SA, Mollee E, Planta RJ. VenemaJ, RaueHA. 1995. E\'olutionarily conser\'ed struCtural elements arc critical for pro­cessing of internal transcribed spacer 2 from .farrbarrJ"!}"ttJ ftrrt1Jiat precursor

ribosomal RNA. J Mol BioI. 250:24-36.

V~lct AI'. DcSalle R. 1994. Evolution and phylogenetic infonnation can·tent of the ITS·1 regioo in the Tiger Beetle Citintltla "mali,. ~lol Bioi E\'ol.1I:29l-405.

\X'esson DM, Poner CH, Collins FH. 1992- Sequence and secondary Struc·ture comparisons of its rDNA in mosquitoes (Diptera: Culicidae). 11101 PhylE\'ol, 1:253-269.

White f\lJD. Dennis £S, Honeycun RI~ Contreras N. 1982. C)10genetics ofthe panhe~icgrasshopper If/amtnlab.t,Trg6 and its bisexual relatives. IX.The ribosomal RJ."lA cistrons. Chromosoma. 85:181-199.

Wilkerson RC. Foster PG. Li C. Sallum MAM. 2005. Molecular phylogeny ofthe ncotropical AnopbtltJ (lVYIJorIrIl1thul) albiklniJ species complex (Diptera:Culicidae). Ann Entomol SCIC Am. 98:918-925.

Wilkerson RC. G.ffigan TV, Lim.J13. 1995. Identification of species relatedto ..'lnopbdtJ (l'{'lJo~",bltJ) albiklniJ by mndom amplified pol)1l'lorphic DNA­Polymerase chain reaction (Diptero: Culicidae). Mem Inst Oswaldo Cruz.90:721-732.

Li and Wilkerson· Anopheles ITS2 Intragenomlc Variation

Wilkerson RC, Parsons TJ, Albright DG, Klein, TA, Braun MJ. 1993. Ran·dom .mplified pol)'Tllorphic DNA {Rt\PD) markers readil)' dislinguish crrP'lie mosquito species (Diptera: Culicidae: Anopbtltl). Insect Mol BioI. I:205-211.

Wilkerson Re, Parsons 1], Klein TA, Gafligan TV, Bergo E, Consolim J.1995. Di:I~:nosis by random amplified pol)1l'lorphic DNA polymerase chainreaction of four cryptic species relaled to Anophtltl (N..JIJo,*,n(bllJ) a/bilanil(Diptera: Culicidae) from Paragu.}', Argentina, and Bt:l7.i1. J Med Entomo!.32:697-704.

\,\'ilkerson Re, Reinen JF. Jj C. 2004. Ribosomal DNA ITS2 sequences dif·ferentiate six species in the Anophtltl mltianl complex (Diptera: Culicid.e).J Med Enotomol. 41:392-401.

Zimmer EA, Manin 51.., Beverly SM, Kan Y"(', Feder JI- 1980. Rapid duoplication and loss of genes coding for the chains of hemoglobin. Proc NatAcad Sci USA. 77:2158-2162-

Zuker M, Mathews DH, Tumer DH. 1999. Algorithms and thennod}'IIamicsfor RNA secondary suucture prediction: a practical guide in RNA biochem­isrry 30d biorechnology. In: Barcis2C\Ooski J, Clark BFC, editors. NATO ASISeries. Dordrecht (Nctherland): K1uwcr Academic Publishen.

Received September 19, 2005Accepted September IS, 2006

Corresponding Editor: Robert Fleischer

59