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This article was downloaded by: [University of Windsor] On: 11 November 2014, At: 06:06 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Aquatic Insects: International Journal of Freshwater Entomology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/naqi20 Cytogenetics of Simulium siamense Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand Pairot Pramual a & Komgrit Wongpakam b a Department of Biology, Faculty of Science , Mahasarakham University , Maha Sarakham, Thailand b Walai Rukhavej Botanical Research Institute , Mahasarakham University , Maha Sarakham, Thailand Published online: 22 Aug 2011. To cite this article: Pairot Pramual & Komgrit Wongpakam (2011) Cytogenetics of Simulium siamense Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand, Aquatic Insects: International Journal of Freshwater Entomology, 33:2, 171-184, DOI: 10.1080/01650424.2011.597407 To link to this article: http://dx.doi.org/10.1080/01650424.2011.597407 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

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Page 1: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

This article was downloaded by: [University of Windsor]On: 11 November 2014, At: 06:06Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Aquatic Insects: International Journalof Freshwater EntomologyPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/naqi20

Cytogenetics of Simulium siamenseTakaoka and Suzuki, 1984 (Diptera:Simuliidae) in northeastern ThailandPairot Pramual a & Komgrit Wongpakam ba Department of Biology, Faculty of Science , MahasarakhamUniversity , Maha Sarakham, Thailandb Walai Rukhavej Botanical Research Institute , MahasarakhamUniversity , Maha Sarakham, ThailandPublished online: 22 Aug 2011.

To cite this article: Pairot Pramual & Komgrit Wongpakam (2011) Cytogenetics of Simuliumsiamense Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand,Aquatic Insects: International Journal of Freshwater Entomology, 33:2, 171-184, DOI:10.1080/01650424.2011.597407

To link to this article: http://dx.doi.org/10.1080/01650424.2011.597407

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Page 2: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

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Page 3: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Cytogenetics of Simulium siamense Takaoka and Suzuki, 1984 (Diptera:

Simuliidae) in northeastern Thailand

Pairot Pramuala* and Komgrit Wongpakamb

aDepartment of Biology, Faculty of Science, Mahasarakham University, Maha Sarakham,Thailand; bWalai Rukhavej Botanical Research Institute, Mahasarakham University, MahaSarakham, Thailand

(Received 18 May 2010; final version received 5 April 2011)

We analysed salivary gland polytene chromosomes of 796 larvae from 17populations of Simulium siamense in northeastern Thailand. Seventeen floatingand two fixed chromosome inversions were recorded. Three cytoforms (A, F andG) were recognised and two of them are new (F and G). Cytoform F isdistinguished by a fixed inversion on the long arm of chromosome II (IIL-8) andcytoform G by fixed inversions on the long arm of chromosome II (IIL-8) andshort arm of chromosome III (IIIS-2). Significant departures from Hardy–Weinberg equilibrium due to heterozygote deficiency in geographically inter-mediate populations and absence of shared polymorphic inversions of thecytoforms indicate separation of the gene pool. Morphometric analysis of thelarvae revealed significant differences in body length (F ¼ 5.00, p ¼0.007) andhead capsule width (F ¼ 4.68, p ¼ 0.010) among cytoforms.

Keywords: black fly; chromosome inversion; cytoform; polytene chromosome;Simulium

Introduction

Salivary gland polytene chromosomes have a crucial role for black fly taxonomy(Adler, Currie and Wood 2004). Cytological studies of the morphological describedspecies have often revealed several cytological sibling species (i.e. species complex)(e.g. Bedo 1979; Tangkawanit, Kuvangkadilok, Baimai and Adler 2009), which canbe recognised by (i) fixed chromosome inversion differences; (ii) sex chromosomedifferences; and (iii) different floating inversions (Rothfels 1956).

Taxonomic knowledge of the black flies in Thailand has increased significantly inthe last decade, with at least 78 morphological species reported. However,cytotaxonomic study is far less developed than morphological taxonomy. Only 16species have been cytologically examined and most of these are simply reports ofstandard polytene chromosome maps. Extensive cytological study of the black fliesin Thailand have included a total of only three species and two species groups,namely S. feuerborni, S. aureohirtum, S. siamense, S. tuberosum species group, S.ceylonicum species group, and also some species of subgenus Gomphostilbia

*Corresponding author. Email: [email protected]

Aquatic Insects

Vol. 33, No. 2, June 2011, 171–184

ISSN 0165-0424 print/ISSN 1744-4152 online

� 2011 Taylor & Francis

DOI: 10.1080/01650424.2011.597407

http://www.informaworld.com

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Page 4: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

(Kuvangkadilok, Phayuhasena and Baimai 1999; Kuvangkadilok, Boonkemtong,Phayuhasena and Baimai 2003; Phasuk, Chanpaisaeng, Adler and Courtney 2005;Jitklang, Kuvangkadilok, Baimai, Takaoka and Adler 2008; Kuvangkadilok,Lualon and Baimai 2008; Pramual, Wongpakam and Kuvangkadilok 2008;Tangkawanit et al. 2009).

Simulium siamense Takaoka and Suzuki, 1984 was morphologically assigned tothe Simulium batoense species group of the subgenus Gomphostilbia (Takaoka andSuzuki 1984). This species has been reported from Nepal, Vietnam and Thailand(Adler and Crosskey 2010). Previous cytological study revealed that S. siamense inThailand is composed of five cytoforms (A, B, C, D and E) (Kuvangkadilok et al.2008). These cytoforms were distinguished by fixed chromosome inversiondifferences (cytoform E), differences in sex chromosomes (cytoform B, C, and D),or differences in floating inversions (cytoform A). In the present study, samplescollected from the less explored region of northeastern Thailand, were cytologicallyexamined. We recognise two additional cytoforms of this species based on fixedchromosome inversion differences. Ecological conditions of the habitats and larvalmorphometrics of the cytoforms are also presented.

Materials and methods

Black fly larvae and pupae were collected from 17 sites in northeastern Thailand(Table 1 and Figure 1). Stream variables including stream width, depth, velocity, pH,water conductivity, water temperature, streambed particle sizes, canopy cover andriparian vegetation were recorded. Larvae were fixed in Carnoy’s solution (3:1, 95%ethanol: glacial acetic acid). Fixative was changed twice within 1 hour and againafter 24 hours. Samples were stored at –208C until chromosome preparations weremade.

Larvaeweremorphologically identifiedusing the key anddescription ofS. siamenseof Takaoka and Suzuki (1984). Penultimate instar larvae were chosen for polytenechromosome preparation. The head and thorax were cut off and kept in 80% ethanolfor further molecular work and the abdomen was used for salivary gland polytenechromosome preparation. Salivary gland polytene chromosomes were prepared usingthe Feulgen method (Rothfels and Dunbar 1953). Polytene chromosomes wereanalysed band for band compared with the standard polytene chromosome map of S.siamense (Kuvangkadilok et al. 2003, 2008) and chromosome rearrangements wererecorded. A test of independence (G test) was use to examine the relationship betweeninversions and sex. TheHardy–Weinberg equilibriumwas assessed using a w2 goodnessof fit test. The cytological relationships of cytoforms of the S. siamense complex wereinferred based on shared chromosomal inversions. Data from a previous study(Kuvangkadilok et al. 2008) were also included in the cytodendrogram.

Last instar larvae, recognised by black gill histoblasts, were used formorphometric analysis. Variability of three morphological characters includingbody length, head capsule width and postgenal cleft width were examined. Bodylength from the top of the head capsule to the posterior circlet was measured usingVernier calipers. Head capsule width represented by the widest point of the headcapsule and postgenal cleft width at the widest point were measured using amicroscope micrometer. One-way analysis of variance (ANOVA) was used to testdifferentiation of the morphological characters among cytoforms and, if significant,post hoc multiple tests were used to determine the differentiation.

172 P. Pramual and K. Wongpakam

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Page 5: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Table

1.

DetailsofthecollectionsitesofSim

ulium

siamense

innortheasternThailand.

Collectionsite

Code

No.oflarvae

analysed

(male:fem

ale)

Latitude/longitude

Altitude

(m)

Date

Cytoform

BanSomsawatUdonThaniProvince

SM1

40(20:20)

178070N/1038350E

230

11Novem

ber

2006

AWangYaiWaterfall,Sisaket

Province

SM55(1)

29(17:12)

148290N/1048320E

202

29September

2007

A,F

SM55(2)

7(4:3)

9Decem

ber

2007

FHuaiNam

Sai,SakonNakhonProvince

SM82

45(37:8)

168550N/1048110E

358

23October

2007

AKangphoWaterfall,MukdahanProvince

SM88

24(8:16)

168440N/1048140E

295

27October

2007

AHuaiKratae,

MukdahanProvince

SM92

78(52:26)

168490N/1048100E

258

27Novem

ber

2007

ABanSamrongkiet,Sisaket

Province

SM95

14(4:10)

148290N/1048310E

215

9Decem

ber

2007

ABanNatambon,Sisaket

Province

SM98

23(15:8)

148260N/1048030E

188

9Decem

ber

2007

AChongSa-ngam,Sisaket

Province

SM99

57(33:24)

148210N/1048030E

314

9Decem

ber

2007

GBanSaeprai,Sisaket

Province

SM100

31(15:16)

148220N/1048040E

290

9Decem

ber

2007

GHuaiLaoWaterfall,Loei

Province

SM128

12(5:7)

178030N/1018420E

346

9February

2008

ABanKamkeaw1,AmnatchareonProvince

SM138(1)

91(54:37)

168100N/1048570E

156

23February

2008

FSM138(2)

77(39:38)

3April2008

FBanKamkeaw2,AmnatchareonProvince

SM139(1)

90(53:37)

168110N/1048550E

160

23February

2008

FSM139(2)

61(36:25)

3April2008

FTheppanaWaterfall,Chaiyaphum

Province

SM146

94(51:43)

158380N/1018250E

613

24February

2008

A,F

HuaiPung,SakonNakhonProvince

SMSK6

23(10:13)

178060N/1038350E

268

2Decem

ber

2007

ATotal

796(453:343)

Aquatic Insects 173

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Page 6: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Results

Chromosome polymorphisms

A total of 796 larvae from 17 populations were cytologically analysed. Nineteeninversions were found (Table 2). Of these, six were previously reported(Kuvangkadilok et al. 2008) and 13 additional inversions were recorded. None ofthese inversions was associated with sex. Most of the inversions were present at a lowfrequency and geographically restricted to a particular population (Table 2). Twoinversions (i.e. IIL-8 and IIIS-2; Figures 2 and 3) were present at high frequency andgeographically widespread (Table 2). Inversion IIL-8 were fixed in seven populationsand almost fixed in two populations (97% in population SM55 and 96% inpopulation SM146) (Table 2). Frequencies of this inversion significantly departedfrom Hardy–Weinberg equilibrium (HWE) in four populations because ofheterozygote deficiencies (Table 3). Inversion IIIS-2 was recorded in eightpopulations (Table 2) where it was fixed in two (SM99 and SM100) and floatingin six other populations. Frequency of IIIS-2 was not in HWE at SMSK6 due toheterozygote deficiency (Table 3).

Cytotaxonomy

Previous study described five cytoforms of S. siamense based on fixed chromosomeinversions differences (cytoform E), sex chromosome differentiation (cytoform B, Cand D), and floating inversions polymorphisms (cytoform A). We found twoadditional cytoforms of this species:

Figure 1. Sampling sites of three cytoforms of Simulium siamense in northeastern Thailand.Circumscribed areas indicate distribution of the three cytoforms. Details of the sampling sitesare presented in Table 1.

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Page 7: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Table

2.

Frequency

ofinvertedconstituents

forinversionsin

threecytoform

sofSim

ulium

siamense.

Cytoform

Population

Larvae

(male

:female)

Inversion

IS-10IS-11IL

-3IL

-4IL

-5IL

-6IIS-1

IIS-2

IIL-1

IIL-5

IIL-7

IIL-8

IIL-9

IIL-10IIIS-2

IIIS-3

IIIL

-3IIIL

-5IIIL

-6

ASM

120:20

00

0.01

0.01

00

0.01

00

0.01

00.28

0.01

00.16

00

00

A,F

SM

55(1)

17:12

00

00

00

00

00

00.97

00

00

00

0F

SM

55(2)

4:3

00

00

00

00

00

01.00

00

00

00

0A

SM

82

37:8

00

00

00

00

00

00.82

00

0.02

00

00

ASM

88

8:16

00

00

00

00

00

00.25

00

0.35

00

00

ASM

92

52:26

00

00

00

00

00

00.49

00

0.37

00

00

ASM

95

4:10

00

00

00

00

00

00.68

00

00

00

0A

SM

98

15:8

00

00

00

0.02

00

00

0.67

0.02

00

00

00

GSM

99

33:24

00.01

00

00

00

00

01.00

00

1.00

00

00

GSM

100

15:16

00

00

00

00

00

01.00

00

1.00

00

00

ASM

128

5:7

00

00

0.04

0.04

00.04

00

00.38

0.04

00.08

0.04

00.17

0F

SM

138(1)

54:37

00

00

00

00

0.01

0.04

01.00

00

00

00

0F

SM

138(2)

39:38

00

00

00

00

00.05

01.00

00

00

00

0F

SM

139(1)

53:37

0.01

00

00

00

00

0.03

0.03

1.00

00

00

00

0F

SM

139(2)

36:25

00

00

00

00

00.06

01.00

00

00

00

0A,F

SM

146

51:43

00

00

00

00

00

00.96

00

00

0.01

00.01

ASMSK

610:13

00

00

00

00

00

00.35

00.02

0.59

00

00

Aquatic Insects 175

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Page 8: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Figure 2. Chromosome arm IIL of Simulium siamense showing IIL-8 homozygous inversion(left) and standard sequence (right).

Figure 3. Chromosome arm IIIS of Simulium siamense showing IIIS-2 homozygousinversion (left) and standard sequence (right).

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Page 9: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Table 3. Tests for Hardy–Weinberg equilibrium of inversions IIL-8 and IIIS-2 in cytoform Aand two populations of cytoform F of Simulium siamense.

Inversion Location

Inversion frequency

ss si ii w2 P

IIL-8 SM1 Obs. 24 11 5 3.71 0.157Exp. 21.66 15.68 2.66

SM55 (1)* Obs. 0 2 32 0.00 1.000Exp. 0.01 1.97 32.01

SM82 Obs. 6 4 35 23.23 50.001Exp. 1.35 13.30 30.35

SM88 Obs. 16 4 4 8.26 0.016Exp. 13.40 9.19 1.40

SM92 Obs. 32 16 30 27.70 50.001Exp. 20.39 39.23 18.39

SM95 Obs. 2 5 7 0.68 0.709Exp. 1.33 6.33 6.33

SM98 Obs. 5 6 13 5.12 0.077Exp. 2.55 10.89 10.55

SM128 Obs. 6 3 3 3.16 0.206Exp. 4.57 5.87 1.57

SM146* Obs. 2 4 88 24.63 50.001Exp. 0.15 7.70 86.15

SMSK6 Obs. 12 6 5 4.64 0.098Exp. 9.67 10.67 2.67

IIIS-2 SM1 Obs. 27 13 0 3.33 0.31Exp. 27.99 11.03 0.99

SM82 Obs. 43 2 0 0.02 0.99Exp. 43.01 1.98 0.01

SM88 Obs. 12 7 5 3.57 0.168Exp. 9.89 11.21 2.89

SM128 Obs. 10 2 0 0.10 0.950Exp. 10.04 1.91 0.04

SMSK6 Obs. 7 5 11 7.60 0.022Exp. 3.80 11.40 7.80

*Populations that are tentatively assigned to cytoform F but inversion IIL-8 was not fixed. Exp., expected;Obs., observed; ss, standard homozygous; si, heterozygous; ii, homozygous inverted.

. Simulium siamense cytoform FCytoform F was characterised by a fixed inversion on the long arm ofchromosome II (IIL-8, Figure 2) and undifferentiated sex chromosomes.Larvae (n ¼ 326) were examined from five populations (SM55(2), SM138(1),SM138(2), SM139(1) and SM139(2)). We also included populations where IIL-8 was present at very high frequency (496%) as cytoform F. Thesepopulations were SM55(1) and SM146. In SM55(1), among 29 larvaeexamined, 27 were homozygous for the IIL-8 inversion and the remainingtwo were heterozygous. For population SM146, 94 larvae were examined and88 were homozyous for IIL-8, four were heterozygous and two were standard.Therefore, it is likely that these two populations belong to cytoform F butsome individuals were cytoform A. Four floating inversions (IS-10, IIL-1, IIL-5 and IIL-7) were recorded. This cytoform was found in varied habitats atelevations ranging from 156 m to 613 m, in small and shallow, slow-flowingstreams with a wide range of riparian vegetation types, canopy cover andstreambed particle sizes (Table 4).

Aquatic Insects 177

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Page 10: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

. Simulium siamense cytoform GCytoform G was characterised by two fixed inversions, IIL-8 (Figure 2) andIIIS-2 (Figure 3), and undifferentiated sex chromosomes. Larvae (n ¼ 88) wereexamined from two populations (SM99 and SM100). A low frequency offloating inversions (IS-11) was recorded for this cytoform. Cytoform G wasfound in habitats at low elevations (230–314 m), in small, shallow, slow-flowing streams with a large streambed particles size (small stones, boulders)and no canopy cover (Table 4).

Cytological relationships among cytoforms

Based on shared chromosome inversions, a cytodendrogram was constructed todepict relationships among seven cytoforms of S. siamense complex in Thailand(Figure 4). Because there are several alternative cytodendrograms the result showingin Figure 4 is one of the hypothesis of relationships. The hypothetical ancestor waspolymorphic for IS-1, IS-5, IS-6, IS-7, IL-1, IIL-1, IIL-2, IIL-5, IIL-7, IIL-8 andIIIS-2 inversions. These inversions continued as polymorphisms, became fixed, orwere lost in the cytoforms.

Six lineages were derived from the hypothetical ancestor. Cytoform A is deriveddirectly from the hypothetical ancestor by 34 floating inversions. Cytoform B arosefrom hypothetical ancestor by IS-2,3 sex-linked inversion (X ¼ IS-2,3). Cytoform Cis derived by the IS-6 sex-linked inversion (X ¼ IS-6) and three floating inversions(IS-1, IS-5, IIIS-2). Cytoform D is derived from hypothetical ancestor by the IS-7sex-linked inversion (X ¼ IS-7) and two floating inversions (IS-1, IIIS-2). CytoformE is derived by two fixed inversions; IS-1 and IIIS-2 and two floating inversions; IL-1and IIL-2. Two cytoforms are derived from a lineage defined by IIL-8 fixedinversion. Cytoform F is derived from this lineage by IS-10, IIL-1, IIL-5, and IIL-7floating inversions and cytoform G is derived by IIIS-2 fixed inversions and IS-11floating inversion.

Morphology

Comparisons of morphological characters revealed significant differences amongthree cytoforms of S. siamense (Table 5). Body length was significant longer incytoform F than in A and G, whereas the latter two were similar. Head capsulewidth was bigger in cytoform A than in cytoform G but neither of them weresignificantly different from cytoform F. Postgenal cleft was not significantly differentamong the three cytoforms.

Discussion

It has been suggested that sibling species are likely to be present in geographicallywidespread species (Adler and McCreadie 1997). Simulium siamense is geographi-cally widespread and found in a wide range of ecological conditions (Kuvangkadiloket al. 2008; Pramual and Kuvangkadilok 2009). This species can be found in mostparts of Thailand and also in Nepal and Vietnam (Adler and Crosskey 2010).Previous cytogenetic study recorded five cytoforms (A, B, C, D, and E) of S.siamense in Thailand.

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Page 11: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Table

4.

Ecologicalconditionsofthelarvalhabitats

ofthreecytoform

sofSim

ulium

siamense

from

northeasternThailand.

AF

G

Stream

variable/cytoform

Min–Max

Mean+

SD

Min–Max

Mean+

SD

Min–Max

Mean+

SD

Altitude(m

)188–358

269.75+

60.52

156–613

235.57+

167.73

290–314

302.00+

16.97

Width

(m)

0.46–1.76

0.93+

0.43

0.44–1.86

0.88+

0.48

0.36–0.55

0.46+

0.13

Depth

(m)

0.03–0.19

0.10+

0.06

0.02–0.51

0.13+

0.17

0.04–0.06

0.05+

0.01

Velocity

(m/s)

0.04–0.96

0.49+

0.32

0.36–0.99

0.56+

0.23

0.57–0.68

0.62+

0.07

Tem

perature

(C8)

20.30–24.10

25.39+

2.50

23.50–28.70

26.14+

2.08

23.30–24.10

23.70+

0.57

pH

6.15–8.13

6.71+

0.68

5.50–7.13

6.49+

0.54

5.70–6.14

5.92+

0.31

Conductivity(mS/cm)

6.00–235.00

49.50+

77.68

12.00–20.00

16.00+

2.70

18.00–20.00

19.00+

1.41

Stream-bed

particle

Sand–Bedrock

Sand–Bedrock

Smallstone–Bedrock

Canopycover

Open–Complete

Open–Partial

Open

Riparianvegetation

Open–Forest

Open–Forest

Forest

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Page 12: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

In the present study, we found two additional cytoforms of this species fromnortheastern Thailand. These cytoforms are distinguished by fixed chromosomeinversion differences (IIL-8 for cytoform F and IIL-8 and IIIS-2 for cytoform G).Inversion IIL-8 was found as a floating inversion in eight populations of cytoform A.A similar situation in which an inversion was fixed in one cytoform and floating inanother has been reported in other black flies such as S. ruficorne (Bedo 1989). Onceit has occurred in a population, an inversion could be lost, fixed or remain as apolymorphism. There are six possible explanations for the spread of the inversions inpopulations according to the fitness effect of the inversions (Hoffmann andRieseberg 2008). Fixation of the IIL-8 inversion in north-eastern populations couldbe due to chance alone (i.e. genetic drift) but this is unlikely to occur simultaneouslyin seven populations. Underdominance is also unlikely because heterozygousindividuals were found at high frequency in several populations. It is alsotheoretically difficult for IIL-8 to increase in a population if it were underdominant(Spirito 1998). Recent theoretical study proposed that the frequency of the inversioncould increase if it captures a pre-existing locally adapted allele (Kirkpatrick andBarton 2006). The inversion then protects the adaptive allele while the inversion itselfincreases in frequency, as the fitness of the individual carrying the inversion increasesfrom the locally adaptive allele. Further study would be interesting to clarify thefitness effect of this inversion.

Figure 4. Cytodendrogram of seven cytoforms of Simulium siamense complex in Thailand.Six lineages are derived from a hypothetical ancestor which polymorphic for IS-1, IS-5, IS-6,IS-7, IL-1, IIL-1, IIL-2, IIL-5, IIL-7, IIL-8 and IIIS-2 inversions. These inversions continuedas polymorphisms, became fixed, or were lost in the cytoforms. Fixed inversions areunderlined, floating inversions are not and sex-linked inversions are indicated as X ¼ IS-2,3 incytoform B, X ¼ IS-6 in cytoform C and X ¼ IS-7 in cytoform D. A line through an inversionmeans that the inversion has been lost.

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Page 13: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

Table

5.

Morphometriccharactersofthreecytoform

sofSim

ulium

siamense

innortheasternThailand.

Character/cytoform

A(n¼

117)

F(n¼

136)

G(n¼

30)

Total

FP

Bodylength

(mm)

Mean+

SD

(range)

4.34+

0.34a

(3.40–5.90)

4.47+

0.37b

(3.90–5.90)

4.35+

0.24a,b

(3.80–4.80)

4.41+

0.35

(3.40–5.90)

5.00

0.007

Headcapsule

width

(mm)

Mean+

SD

(range)

617.44+

40.92a

(490–750)

581.67+

40.26a,b

(520–710)

597.67+

25.96b

(550–650)

610.39+

36.42

(490–750)

4.68

0.010

Postgenalcleftwidth

(mm)

Mean+

SD

(range)

194.27+

20.69

(150–300)

196.40+

17.41

(170–280)

195.67+

17.36

(160–230)

195.44+

18.80

(150–300)

0.40

0.669

Post

hoctest

usingTukey

HSD;thesamesubscriptletter

indicatesnosignificantdifference

atp-valueof0.05.nrepresents

thenumber

oflarvaeanalysed.

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Page 14: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

The IIL-8 inversion showed significant departure from Hardy–Weinbergequilibrium (HWE) in three populations (SM82, SM88, SM92) that weregeographically intermediate between cytoform A and cytoform F (Figure 1).Heterozygote deficiency could be due to a mixture of genetically distinct populationsarising through migration, selection against the heterozygote or assortative matingwithin cytoforms (Bedo 1979). Selection against heterozygotes occurs if the inversionhas an underdominance effect. However, this is unlikely because if the IIL-8 wasunderdominant, we should not expect to see the heterozygous individuals at highfrequency in several populations. Migration is also unlikely given that highfrequency of the IIL-8 heterozygote was found in cytoform A. The most likelyexplanation is non-random mating. This would increase homozygous and decreaseheterozygous individuals and thus a heterozygous deficiency will occur.

Heterozygous deficiency in the geographically intermediate populations indicatedlimitation of gene flow between cytoforms. This is also supported by a uniquefloating inversion in each cytoform, although most inversions were present at lowfrequency and absence of these inversions in particular populations could be due tochance alone. However, inversion IIIS-2 was present at high frequency (2–59%) incytoform A but was absent from cytoform F. Limitation of gene flow betweencytoforms and heterozygote deficiency in geographically intermediate populationsmight indicate a separation of the gene pool between these cytoforms.

Cytoform G was found in two populations. This cytoform is closely related tocytoform F because they share the fixed IIL-8 inversion. Cytoform G is also relatedto cytoform E, as both shared the IIIS-2 fixed inversion. Cytoform G wasgeographically intermediate between cytoforms F and E. The fixed inversions thatdistinguish this cytoform are also intermediate between cytoforms F and E.Inversion IIL-8 was fixed in cytoform F and IIIS-2 was fixed in cytoform E.Therefore, cytoform G might be derived from interbreeding between cytoforms Fand E. Alternatively, inversions distinguishing these cytoforms are shared ancestralinversions with different fates in different lineages.

Cytoforms G and F were geographically adjacent, with less than 50 kmseparating them. No IIIS-2 was found in cytoform F, although this inversion wasfound in seven populations of cytoform A. Absence of the IIIS-2 inversion incytoform F indicated that no gene flow occurred between these cytoforms. Giventhat geographic separation of the two cytoforms is close compared to the dispersalability of black flies (Crosskey 1990), the absence of gene flow between thesecytoforms might indicate separation of the gene pool.

Populations tentatively assigned to cytoform A in this study seem to be differentfrom cytoform A reported by Kuvangkadilok et al. (2008). In the present study,populations that were assigned to cytoform A were characterised by a lack of fixedchromosome inversions or sex-linked inversions. These populations, however, have ahigh frequency of some floating inversions: inversion IIL-8 was found at up to 82%and inversion IIIS-2 was found at up to 59%. In contrast, Kuvangkadilok et al.(2008) reported that cytoform A has a high frequency of IS-1 and IIIS-1, which areabsent in the populations of our cytoform A. Because the geographic distribution ofcytoform A is large, covering most of Thailand, different inversion frequencies indifferent populations might reflect local adaptation. It is probable that cytoform A isactually composed of multiple cytoforms.

Sibling species often have similar morphology and can be structurallyindistinguishable. Some studies, however, report diagnostic structural characters

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Page 15: Cytogenetics of               Simulium siamense               Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

for sibling species. For example, Jitklang et al. (2008) reported diagnostic charactersfor the Simulium ceylonicum species group. Morphological comparison revealedsignificant differences in body length and head capsule width among cytoforms butas the ranges of these characters overlapped, they could not be used as diagnosticcharacters for cytoform identification. Further study using molecular geneticmarkers would be helpful for cytoform identification.

Ecological study in several black fly species revealed the association betweencytological sibling species and ecological conditions of the larval habitats (Adler andMcCreadie 1997). Kuvangkadilok et al. (2008) noted that cytoforms B, C and Doccurred in habitats with high levels of dissolved oxygen, water conductivity andwater temperature. Cytoforms A and E were found in large and fast-flowing streamswith low water conductivity. Ecological observations revealed that cytoform A, Fand G share similar larval habitats with largely overlapping ranges of streamvariables. However, we noted that cytoform F and G were found more often in openhabitats than cytoform A.

In conclusion, our results indicate that even within species that have been wellstudied, we could still find new cytoforms. This suggests that there is still hiddenbiodiversity in black flies in Thailand. Development of molecular genetic markersfor cytoform identification would be useful. Results also indicate the importantrole of chromosome inversions in local adaptation and evolution of black flies.Further study, especially the development of molecular markers to study thenucleotide region inside chromosome inversions, could be particularly useful.

Acknowledgements

This work was financially supported by grant from Mahasarakham University (grant year2010). We would like to thank Peter Adler (Clemson University, USA) and Adrian Plant(National Museum of Wales, UK) for valuable comments on the manuscript.

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