are black ﬂies of the subgenus wilhelmia (diptera: simuliidae) multiple ... · are black ﬂies...

Are black flies of the subgenus Wilhelmia (Diptera:Simuliidae) multiple species or a single geographicalgeneralist? Insights from the macrogenome

PETER H. ADLER1*, ABDULLAH INCI2, ALPARSLAN YILDIRIM2, ONDER DUZLU2,JOHN W. MCCREADIE3, MATÚŠ KÚDELA4, ATEFEH KHAZENI5,TATIANA BRÚDEROVÁ4, GUNTHER SEITZ6, HIROYUKI TAKAOKA7,YASUSHI OTSUKA8 and JON BASS9

1Entomology Program, Clemson University, Clemson, SC 29634-0310, USA2Department of Parasitology, Faculty of Veterinary Medicine, Erciyes University, Kayseri, Turkey3Department of Biological Sciences, University of South Alabama, Mobile, AL 36688, USA4Department of Zoology, Comenius University, SK 842 15 Bratislava, Slovakia5Department of Parasitology and Mycology, Isfahan University of Medical Sciences, Isfahan73461-81746, Iran6District Government of Lower Bavaria, Regierungsplatz 540, 84028 Landshut, Germany7Institute of Biological Sciences, University of Malaya, Kuala Lumpur 50603, Malaysia8Research Center forPacific Islands,Kagoshima University,Korimoto 1-21-24,Kagoshima,890-8580,Japan9The Wessex Chalk Stream and Rivers Trust, Kimbridge, SO51 0LE, UK

Received 21 June 2014; revised 9 August 2014; accepted for publication 9 August 2014

Organisms with vast distributions often represent geographical mosaics of cryptic species. The black fly Simulium(Wilhelmia) lineatum is among the most widely distributed members of the family Simuliidae, ranging from theBritish Isles to eastern China. Rather than viewing S. lineatum as a possible aggregate of multiple species,taxonomists have suggested a more inclusive taxon with additional synonyms. Accordingly, S. lineatum is an idealcandidate for testing the hypothesis that a wide geographical distribution signals the presence of more than onespecies. A cytogenetic approach was used to probe the macrogenome of S. lineatum and other taxa proposed bytaxonomists as conspecific. The banding patterns in the polytene chromosomes of 480 larvae from 15 countries acrossthe Palearctic Region revealed 128 rearrangements of the complement. All rearrangements were autosomal and 89%were inversions nonrandomly distributed among species and among chromosome arms. The analyses clarifylong-standing confusion over previously proposed names and reveal a longitudinal succession of four speciessequentially replacing one another from west to east: Simulium lineatum s.s., Simulium balcanicum, Simuliumturgaicum, and Simulium takahasii. Thus, S. turgaicum is recalled from synonymy and the other three species arevalidated. Within the most-represented species, S. balcanicum, the frequency of inversions follows a longitudinalgradient with a north–south bias; as the distance between the sites increases along this north-west–south-east axis,the similarity of inversion frequencies between sites decreases. Validation of the concept that broadly distributedblack flies are composites of structurally similar species provides a framework for guiding discovery of additionalbiodiversity. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114, 163–183.

ADDITIONAL KEYWORDS: biodiversity – pests – polytene chromosomes – Simulium lineatum.

INTRODUCTION

Much of ecology and applied biology is focused atthe species level: from understanding the effects of

specialization on metacommunity dynamics (Pandit,Kolasa & Cottenie, 2009) to controlling the vectors ofdisease agents and managing pests (Adler, 2009;Kamali et al., 2012). Species perceived as generalists,however, often involve cases of mistaken identity;putative species with broad host or geographical*Corresponding author. E-mail: [email protected]

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Biological Journal of the Linnean Society, 2015, 114, 163–183. With 14 figures

© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114, 163–183 163

mailto:[email protected]

ranges tend to be composites of structurally similar(cryptic) species, each occupying a discrete subset ofthe larger range (Williams et al., 2012; Hambäcket al., 2013).

The family Simuliidae, through study of the giantpolytene chromosomes in the larval silk glands, pro-vides classic examples of cryptic biodiversity in taxaonce considered single widespread species occupyingdiverse habitats (Rothfels, 1979; Adler, Cheke & Post,2010). Current examples of generalists in theSimuliidae are found in the Palearctic Simuliumsubgenus Wilhelmia. Among the 26 species in thesubgenus are some of the most widely distributedblack flies in the Old World (Adler & Crosskey,2014). Simulium lineatum (Meigen) as currently rec-ognized, for example, extends from southern Spain(Gallardo-Mayenco & Toja, 2002) and the British Islesacross a vast swath of the Palearctic through the Altaisouth to Iran and eastward to the coast of China, adistance of more than 9500 km (Adler & Crosskey,2014). Although wide distributions typically signal thepresence of cryptic species (Adler et al., 2004, 2010),S. lineatum has been viewed as broadly cohesive, andtwo former species, Simulium salopiense Edwards ofWestern Europe and Simulium turgaicum Rubtsovof Central Asia, were declared conspecific withS. lineatum (Crosskey & Davies, 1972; Crosskey &Howard, 1997). Populations of Wilhelmia in Japan andKorea were considered conspecific with S. lineatumuntil 1962, when they were described under the nameSimulium takahasii (Rubtsov), although this wasbased on questionable structural differences. Morerecently, Simulium balcanicum (Enderlein), whichdiffers structurally only in the pupal stage by havinga petiolate pair of gill filaments, was posited asconspecific with S. lineatum (Crosskey & Zwick, 2007).

The chromosomes of seven nominal species of thesubgenus Wilhelmia have been described (Grinchuk &Chubareva, 1974; Knoz, Grinchuk & Chubareva,1976; Petrova et al., 2003; Chubareva, Petrova &Reva, 2007; Chubareva & Petrova, 2008; Huang et al.,2012). Only one study, however, attempted a bandingcomparison between species [Simulium equinum (L.)and S. lineatum in south-western Germany (Weber &Grunewald, 1989)] and only one study, althoughlimited to Armenia, considered the possibility ofcryptic species (in Simulium paraequinum Puri)(Petrova et al., 2003). All chromosomally knownmembers of the subgenus Wilhelmia share a whole-arm interchange (IS + IIL, IL + IIS) possibly homolo-gous with that in the African subgenus Metomphalus(Vajime & Dunbar in Rothfels, 1979). The only otherexamples of a IS + IIL, IL + IIS interchange in theSimuliidae (Rothfels & Freeman, 1983; Procunier,1982) are of independent origin in distantly-relatedtaxa (i.e. different genera).

Our initial motivation for a cytogenetic study ofWilhelmia was sparked by a severe outbreak of blackflies that affected tourism and livestock productionalong the Kizilirmak River in Turkey’s CappadociaRegion during the middle of the first decade of 2000(Yilmaz et al., 2007; Sariözkan et al., 2014). To deter-mine the species involved in the Cappadocia outbreak,we conducted a cytogenetic analysis of the populationof Wilhelmia in the Kizilirmak River. To reconcile theoutbreak with the pest status of the relevant species inother regions and to place our results in an interpre-tive context, we adopted a collaborative approach andexpanded the geographical coverage to include theentire Palearctic Region. Our primary objective was totest the hypothesis that S. lineatum is a single species,as some taxonomists have claimed, versus a compositeof one or more of the variously recognized species orsynonyms: S. balcanicum, S. lineatum s.s., S. salopi-ense, S. takahasii, and S. turgaicum. We examinedmaterial from populations typically regarded as rep-resentative of each of these names.

MATERIAL AND METHODSCOLLECTION OF MATERIAL

Larvae were hand collected from trailing vegetationat 18 sites in 15 countries (Table 1, Fig. 1) and fixedin two or three changes of modified Carnoy’s solution(1 : 3 acetic ethanol). Larvae patently infected withparasites (morphologically identified) were recorded(Tables 2, 3). Larval carcasses and photographic nega-tives of chromosomes were deposited in the ClemsonUniversity Arthropod Collection, Clemson, SC.

CHROMOSOME PREPARATION

Polytene chromosomes were prepared using theFeulgen technique of Rothfels & Dunbar (1953), inaccordance with the procedures outlined by Adler &Huang (2011), although modified by using acold, 30-min treatment in 5 N hydrochloric acid(Charalambous et al., 1996). The silk-gland contentsof larvae typically solidified into a gelatinous masswithin as little as a week of fixation; thus, afterFeulgen staining, the chromosomes were strippedfrom the mass with fine needles. Gender was deter-mined by gonadal shape. The ovoid to spherical malegonads of the subgenus Wilhelmia are among thelargest in the Simuliidae; their large size and melaninsheath made gender determination straightforward.Female gonads were elongated and ensheathed withmelanin only at the distal end.

CHROMOSOME INTERPRETATION

Selected chromosomes from larvae collected in Tur-key’s Kizilirmak River in September and October 2011

164 P. H. ADLER ET AL.

© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114, 163–183

Tab

le1.

Col

lect

ion

sof

Sim

uli

um

(Wil

hel

mia

)sp

ecie

sfr

omE

uro

pean

dA

sia

Sit

eL

ocat

ion

Lat

itu

de,

lon

gitu

deE

leva

tion

(ma.

s.l.)

Dat

eS

peci

es

1A

rmen

ia,

Kot

ayk

Pro

vin

ce,

Hra

zdan

,H

razd

anR

iver

,be

low

rese

rvoi

r40

°30′

31″N

44°4

4′23

″E16

959

Jun

e19

99S

imu

liu

mtu

rgai

cum

2A

ust

ria,

Sto

pfen

reu

th,

side

chan

nel

ofD

anu

beR

iver

48°0

8′43

″N16

°53′

26″E

140

8M

ay20

11S

imu

liu

mba

lcan

icu

m

3F

ran

ce,

Cor

sica

,C

alvi

ani,

Tavi

gnan

oR

iver

42°0

7′02

″N09

°29′

26″E

617

Sep

tem

ber

2013

Sim

uli

um

lin

eatu

m

4G

erm

any,

Ru

hst

orf,

Rot

tR

iver

48°2

5′44

″N13

°20′

01″E

315

3M

arch

2012

Sim

uli

um

balc

anic

um

,S

imu

liu

mli

nea

tum

5G

erm

any,

nea

rL

ands

hu

t,Is

arR

iver

48°3

0′31

″N12

°04′

23″E

401

11M

arch

2012

Sim

uli

um

lin

eatu

m

6G

reec

e,A

liak

mon

as,

Ali

akm

onas

(=H

alia

cmon

)R

iver

40°1

8′10

″N21

°25′

59″E

550

3M

ay20

13S

imu

liu

mba

lcan

icu

m

7Ir

an,

Isfa

han

,N

azh

van

,Z

ayan

deh

rood

Riv

er32

°38′

26″N

51°3

7′58

″E15

8026

Mar

ch20

11S

imu

liu

mtu

rgai

cum

8It

aly,

San

Pao

lo,

Tagl

iam

ento

Riv

er45

°53′

16″N

12°5

6′21

″E15

22S

epte

mbe

r20

13S

imu

liu

mli

nea

tum

9Ja

pan

,O

ita

Pre

fect

ure

,Y

ufu

,Y

ufu

in,

trib

uta

ryof

Oit

aR

iver

33°1

5′51

″N13

1°22

′02″

E46

510

Dec

embe

r20

13S

imu

liu

mta

kah

asii

10M

aced

onia

,D

emir

Kap

ija,

Var

dar

Riv

er41

°24′

24″N

22°1

6′14

″E10

02

May

2013

Sim

uli

um

balc

anic

um

11P

olan

d,S

tary

Sac

z,D

un

ajec

Riv

er49

°34′

57″N

20°3

7′58

″E29

014

Jun

e20

13S

imu

liu

mli

nea

tum

12R

oman

ia,

Sim

eria

Vec

he,

Str

eiR

iver

45°5

0′09

″N23

°02′

48″E

190

17S

epte

mbe

r20

12S

imu

liu

mba

lcan

icu

m

13R

oman

ia,

Bâr

za,

Cer

na

Riv

er44

°50′

26″N

22°2

3′15

″E11

025

Sep

tem

ber

2011

Sim

uli

um

balc

anic

um

14R

oman

ia,

Bai

leH

ercu

lan

e,C

ern

aR

iver

44°5

4′05

″N22

°25′

47″E

180

12A

pril

2012

Sim

uli

um

balc

anic

um

15S

erbi

a,V

elik

aP

lan

a,V

elik

aM

orav

aR

iver

44°2

0′26

″N21

°07′

25″E

901

May

2013

Sim

uli

um

balc

anic

um

16S

lova

kia,

Mu

žla,

Dan

ube

Riv

er47

°46′

05″N

18°3

2′19

″E10

213

May

2010

Sim

uli

um

balc

anic

um

17Tu

rkey

,K

izil

irm

akR

iver

*38

°N34

°E92

520

10–2

012

Sim

uli

um

balc

anic

um

,S

imu

liu

mtu

rgai

cum

18U

nit

edK

ingd

om(E

ngl

and)

,E

ast

Sto

ke,

Riv

erF

rom

e50

°40′

56″N

02°1

0′59

″W7

6A

pril

2013

Sim

uli

um

lin

eatu

m(a

sS

imu

liu

msa

lopi

ense

)

*Giv

enth

epr

oxim

ity

ofco

llec

tion

s(w

ith

inap

prox

imat

ely

20km

ofon

ean

oth

er),

smal

lsa

mpl

es(S

.bal

can

icu

m),

and

min

imal

poly

mor

phis

ms

(S.t

urg

aicu

m),

chro

mos

omal

data

are

com

bin

edin

Tabl

es2

and

3fo

r4

site

son

the

Kiz

ilir

mak

Riv

er:A

van

os(3

8°42

′57.

30″N

34°5

0′27

.50″

E),

925

m,

18O

ctob

er20

10;

Gü

lseh

irB

ridg

e,38

°45′

22.2

6″N

34°3

7′01

.15″

E,

905

m,

13O

ctob

er20

11;

Gü

lseh

ir-H

acib

aekt

asR

oad,

38°4

5′38

.30″

N34

°36′

39.2

9″E

,90

4m

,13

Oct

ober

2011

;an

dG

üls

ehir

,38

°45′

25.1

7″N

34°3

6′55

.32″

E,

904

m,

24S

epte

mbe

r20

11an

d15

Jan

uar

y20

12.

MACROGENOME OF BLACK FLIES IN SUBGENUS WILHELMIA 165


and in Japan in December 2013 were photographedunder oil immersion on an Olympus BX40 compoundmicroscope. Maps (Figs 2, 3, 4, 5, 6, 7, 8, 9, 10) wereprepared using PHOTOSHOP ELEMENTS, version 8(Adobe Inc.). The entire complement (chromosomes I,II, and III) was divided into 100 arbitrary sections ofapproximately equivalent length, beginning with theshort arm (S) of I and continuing through the long arm(L) of III. We maintained the section limits of Weber &Grunewald (1989) wherever possible, although ourattempts were stymied by the low resolution of theirmaps and the presence of heterozygous inversions(e.g. in the middle of IS) and geographically local,homozygous inversions (e.g. in the base of IIL) on theirmaps. For example, the IL, IIL, and IIIL photomaps forS. lineatum of Weber & Grunewald (1989) carry theIL-10, 11 and IIL-8, 9, 14, and IIIL-5 homozygoussequences, respectively, of our system, and IS in theirphotomap is heterozygous for our IS-4 sequence. Wetherefore established the standard map de novo, basedon the most common sequence in each arm across oursampled populations. Chromosomal rearrangements,chiefly inversions and heterobands, are indicated bybrackets or arrows on our maps. Inversions typicallywere numbered in order of their discovery. Eachheteroband (hb) was named for the section in which it

occurred (e.g. IIIL 97hb), as were supernumerarybands (e.g. IIIL 99i). Fixed inversions across all sites ofa species are italicized.

Frequencies of all polymorphisms, based on indi-vidual chromosomal homologues, were calculated(Tables 2, 3). Twenty-four (4.8%) of 504 chromosomallyprepared larvae could not be compared completelywith the standard map and were excluded from calcu-lations of polymorphism frequencies and all otherevaluations. Fifteen of these larvae were from Europe’slate-summer generation (September) of minusculemature larvae (length, mean ± SE = 5.3 ± 0.07 mm,N = 14) of S. lineatum s.s. The remaining nineindividuals (all S. balcanicum) with chromosomesnot amenable for analysis were pharate pupae.Polymorphisms of sufficient frequency were tested forHardy–Weinberg equilibrium. We used chi-squaredtests to evaluate independence of inversion sequencesand gender, and goodness of fit tests to determine if thedistribution of inversions was equal among species andamong chromosome arms.

GILL MORPHOLOGY

The lengths of all eight gill filaments (Fig. 11) weremeasured using IMAGEJ (NIH) for representative

Figure 1. Map of collecting sites for four species of Simulium (Wilhelmia) in the Palearctic Region; numbers correspondto sites in Table 1.



Table 2. Frequency of chromosomal rearrangements in Simulium balcanicum from Austria (Site 2), Germany (Site 4),Greece (Site 6), Macedonia (Site 10), Romania (Sites 12, 13, 14), Serbia (Site 15), Slovakia (Site 16), and Turkey (Site 17),2010–2013

Site 2 4* 6 10* 12 13 14 15 16 17

Females : Males 6 : 1 21 : 7 1 : 0 18 : 15 18 : 10 5 : 2 0 : 1 22 : 12 7 : 2 10 : 7IS-2 0.03IS-4 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00IS-8 0.02IS-9 0.06IS-18 0.02IL-1 0.79 0.93 0.47† 0.61‡ 0.43 0.49† 0.89 0.62IL-11 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00IL-13 0.29 0.16 0.12 0.23† 0.07 0.07 0.33IL-14 1.00 0.97 1.00 1.00 1.00 1.00 1.00 0.99 1.00 1.00IL-15 0.04IL-16 0.06IIS-1 0.29 0.27 0.50 0.39† 0.34 0.29 0.50 0.32† 0.33 0.68IIS-2§ 0.03IIS-3§ 0.02 0.03IIS-4 0.03IIS-5 0.02 0.03IIS-6 0.14 0.04 0.02 0.09 0.06 0.06 0.09IIS-7 0.03IIS-8 0.03IIS-9§ 0.03IIS-10§ 0.03IIS-11 0.03IIS-12 0.03IIS-13 0.03IIS-15 0.04 0.07IIS-16 0.06 0.04 0.03IIS-17 0.04 0.03 0.02 0.14 0.01 0.06IIS-18 0.02IIS-19 0.03IIS-20§ 0.02 0.02 0.50IIS-21 0.02IIS-22 0.03IIS-23 0.02IIS-24 0.50 0.05 0.04IIS-26§ 0.02IIS-27 0.02IIS-28 0.01IIS-29 0.04 0.03IIS-30 0.04 0.03IIS-31§ 0.01IIS-32 0.02IIS-complex§¶ 0.02 0.06 0.06IIS 54hb 0.07IIL-1 0.79 0.95 1.00 0.82 0.96 0.93 1.00 0.94 0.78 1.00IIL-2 0.32IIL-4 0.29IIL-6 0.03IIL-7 0.07 0.04 0.05 0.06 0.32IIL-8 0.17 0.04 0.07 0.04IIL-11 0.07 0.02 0.06 0.04 0.03 0.17IIL-12 0.07 0.02 0.09 0.04 0.03IIIL-1 0.07 0.13 0.02 0.07 0.04 0.06 0.03IIIL-7 0.02 0.06IIIL-8 0.07 0.05IIIL-9 0.93 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00IIIL-10 0.93 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00IIIL-11 0.02IIIL-12 0.11 0.07IIIL-13 0.06IIIL-15 0.04 0.01IIIL 97hb 0.07IIIL 100hb1 0.02Mean no. heterozygous autosomal inversions/larva 2.29 1.02 2.00 2.42 2.39 2.14 2.00 0.99 3.00 3.77

*Site 4 : one male larva was infected with the microsporidium Polydispyrenia simulii. Site 10 : one female larva was infected with a mermithid nematode.†The following inversions were in Hardy–Weinberg equilibrium (d.f. = 1, P > 0.05): IL-1 at Site 10 (ss = 10, si = 15, ii = 8, where s = standard and i = inverted; χ2 = 0.25) andSite 15 (ss = 11, si = 13, ii = 10; χ2 = 1.87); IL-13 at Site 12 (ss = 18, si = 7, ii = 3; χ2 = 2.50); IIS-1 at Site 10 (ss = 11, si = 18, ii = 4; χ2 = 0.67) and Site 15 (ss = 15, si = 16,ii = 3; χ2 = 0.19).‡IL-1 was not in Hardy–Weinberg equilibrium (ss = 7, si = 8, ii = 13; χ2 = 4.50, d.f. = 1, P < 0.05).§The inversion occurred on top of the IIS-1 sequence.¶The breakpoints of overlapping heterozygous inversions on top of a IIS-1 homozygote were not resolved in three larvae; the unresolved inversions at the 3 different sites(12, 16, and 17) were not necessarily the same inversion.



Table 3. Frequency of chromosomal rearrangements in three species of Simulium (Wilhelmia) from Armenia (Site 1),France (Site 3), Germany (Sites 4, 5), Iran (Site 7), Italy (Site 8), Japan (Site 9), Poland (Site 11), Turkey (Site 17), andEngland (Site 18), 1999 and 2010–2013

Site

Simulium lineatum Simulium takahasii Simulium turgaicum

3 4 5* 8 11 18* 9 1 7* 17*

Females : Males 5 : 7 0 : 1 9 : 10 4 : 2 1 : 0 31 : 18 19 : 30 1 : 0 22 : 12 82 : 61

IS-1 0.01IS-3 0.03IS-4 1.00 0.76 1.00 0.50 1.00IS-5 0.05 0.06IS-6 1.00 0.32 1.00 0.50 0.16†IS-7 0.03 0.08IS-10 0.04IS-11 0.02IS-12‡ 0.07IS-13‡ 0.07IS-14‡ 0.07IS-15‡ 0.07IS-16‡ 0.07IS-17 0.01IS N.O.§ 0.01IL-2 < 0.01IL-3a¶ < 0.01IL-3b¶ 0.32†IL-4 < 0.01IL-5 < 0.01IL-6 < 0.01IL-7 < 0.01IL-8 < 0.01IL-9 < 0.01IL-10 0.88 0.84 1.00 1.00IL-11 0.96 0.55 1.00 0.98IL-12 0.03IL-17 0.39†IL-18 0.24†IL-19 0.50IL-20 0.04IL-21 0.01IL 29hb 1.00IL 34hb 0.13IL 35hb 0.06IL 41hb < 0.01IL 29hb 1.00IIS-14 0.08 0.42IIS-25 0.50IIS 45i 0.01IIL-3 0.05IIL-5 < 0.01IIL-8 0.58 1.00 0.66† 1.00 0.39†IIL-9 1.00 0.50† 1.00 0.81IIL-10 0.21 0.08IIL-11 0.58 0.50 0.66 0.83 1.00 0.35†IIL-12 0.29IIL-13 0.03IIL-14 0.16 0.08 0.08IIL-15** 0.07IIL-16** 0.07IIL-17 0.01IIL-18 0.01



pupae of S. balcanicum from Site 2 (N = 2); S. linea-tum from Sites 3 (N = 7), 8 (N = 3), and 18 (N = 1);S. takahasii from Beijing, China (N = 3); andS. turgaicum from Sites 7 (N = 3) and 17 (N = 5).Principal components analysis was used to displaydifferences in gill measurements including the ratiosof filaments 5 : 2 and 3 : 1 in multidimensional space.

STATISTICAL ANALYSIS OF INVERSION FREQUENCIES

Only S. balcanicum was collected in adequatenumbers (7–34 larvae/site) over a sufficient number ofsites (N = 8) for analysis of inversion frequencies.Samples were collected across north–south and east–west gradients (Table 1). These gradients represent

not only changes in location, but also measures ofdistance. Correlations were therefore used to comparethe relationships of distance to degrees north andeast. These correlations were based on all pairwisecomparisons of sites, rendering the data inherentlynon-independent; significance tests therefore werebased on 1000 random permutations (Lunneborg,2000). Regression analysis was used to determinewhether the distance gradients between sites haddirectionality. For example, are sites in more northernlocations also found in western, as opposed to eastern,locations? Because there is no predictor or responsevariable in this case, the regression equation wascomputed using reduced major-axis regression (Sokal& Rohlf, 1995), with degrees north arbitrarily assigned

Table 3. Continued

Site

Simulium lineatum Simulium takahasii Simulium turgaicum

3 4 5* 8 11 18* 9 1 7* 17*

Females : Males 5 : 7 0 : 1 9 : 10 4 : 2 1 : 0 31 : 18 19 : 30 1 : 0 22 : 12 82 : 61

IIL-19†† 0.02IIL-20 0.01IIL-21 0.01IIL 63hb 0.08IIIS-1 0.01IIIS-2 0.13 0.01IIIS-3 1.00IIIS-4 0.02IIIS 77hb < 0.01IIIL-2 < 0.01IIIL-3 < 0.01IIIL-4 < 0.01IIIL-5 1.00 0.50 0.96 1.00 1.00 1.00IIIL-6 0.50 0.03IIIL-10 1.00IIIL-14 0.07IIIL-complex 0.50 0.05IIIL 99i 1.00IIIL 100hb2 0.01Mean no. heterozygous autosomal inversions/larva 1.42 5.00 4.68 1.33 4.00 4.31 0.12 0.00 0.03 0.12

*Site 5 : 1 male larva was infected with the microsporidium Amblyospora sp., and 1 male larva was infected with a mermithid nematode. Site 18:one male larva was infected with Amblyospora varians. Site 7: one male larva was infected with Amblyospora bracteata. Site 17: one male larvawas infected with the microsporidium Polydispyrenia simulii; one male larva was infected with Amblyospora bracteata, and breakpoints of a mid-ISheterozygous inversion were not determined for this individual.†The following inversions were in Hardy-Weinberg equilibrium (d.f. = 1, P > 0.05): IS-6 at Site 18 (ss = 35, si = 12, ii = 2, where s = standard andi = inverted; χ2 = 0.53); IL-3b at Site 18 (ss = 24, si = 18, ii = 7; χ2 = 1.33); IL-17 at Site 18 (ss = 21, si = 18, ii = 10; χ2 = 2.51); IL-18 at Site 18 (ss = 31,si = 14, ii = 5; χ2 = 2.70); IIL-8 at Site 5 (ss = 4, si = 5, ii = 10; χ2 = 3.28) and Site 18 (ss = 17, si = 26, ii = 6; χ2 = 0.68); IIL-9 at Site 5 (ss = 5, si = 9,ii = 5; χ2 = 0.05); IIL-11 at Site 18 (ss = 21, si = 22, ii = 6; χ2 = 0.00).‡Five inversions (IS-12, IS-13, IS-14, IS-15, and IS-16) were completely linked and paired with the standard homologue in seven larvae (six females,one male).§Heterozygous for nucleolar organizer (N.O.) expression.¶Although IL-3a and IL-3b have the same macro-breakpoints, they must be independent inversions because IL-3b occurred only if IL-10 waspresent, whereas IL-3a occurred without IL-10.**IIL-15 and IIL-16 were completely linked and were found in all, and only, the same seven larvae with linked inversions IS-12, IS-13, IS-14, IS-15,and IS-16.††IIL-19 occurred only on top of IIL-9.



to the left side of the equation. A distance–decayrelationship (DDR) between site similarity, based oninversion-frequency profiles, and Euclidean distancebetween sites was examined using linear regression(Soininen, McDonald & Hillebrand, 2007). Similaritybetween sites was calculated as the Bray–Curtisresemblance coefficient of the square-root transformedfrequency of inversions (McCune & Grace, 2002). Weestablished a priori that the frequency of an inversionhad to be greater than zero for at least two of the eightsites to be included in the analysis. Significance testsof the DDR were based on 1000 random permutations(Lunneborg, 2000). Both similarity and distance wereexpressed on a log-log scale for this regression. Bydefinition, when a site is compared to itself, it will have100% similarity in the inversion profile and zerodistance, which could increase the overall strength ofthe DDR unfairly. Accordingly, we used a trimmed dataset in which these values were removed. Relationshipsbetween individual inversions and degrees north andeast were examined with simple linear regression(log-log scale).

RESULTSGENERALITIES

The characteristic Wilhelmia whole-arm interchange(IS + IIL, IL + IIS) was present in all larvae. Thecentromeres of the three metacentric chromosomeswere associated tightly in a persistent chromocentrein all nuclei. Although additional heterochromatinthat would suggest a genuine chromocentre (Fig. 2)was not evident in the majority of populations, it waspresent, although minimal, in some populations (e.g.

Site 10). Ectopic pairing of telomeres occurred insome populations of S. balcanicum (e.g. Site 15) andS. lineatum (e.g. Sites 3 and 11). The telomeres, espe-cially of IIS and, to a lesser extent IIIS (Fig. 8B), ofS. takahasii typically had extra heterochromatin.Sticky heterochromatin in IIIL, manifesting as bandsadhering to one another, particularly in section 92(Fig. 10B), was typical of populations at some sites(e.g. S. balcanicum at Site 10, S. turgaicum at Site17, and, less frequently, S. takahasii at Site 9).

Classic chromosomal markers that are widespreador universal in the Simuliidae (Rothfels, Feraday& Kaneps, 1978), such as the parabalbiani, werepresent in all material (Figs. 2, 3, 4, 5, 6, 7, 8, 9, 10).The doublet marker (dt; Fig. 7), a conspicuoussubterminal pair of bands in IIL (section 71),although taxonomically more restricted, minimally tothe subgenus Wilhelmia, provided utility as an addi-tional marker. The nucleolar organizer was in thebase of IS (Figs 2, 3). The bulge in IIS was cleaved bythe ring of Balbiani (Fig. 6). The end of IIIS wastypically flared, obscuring the banding in section 72.Clusters of chromatids could be discerned in theflared region at some sites (e.g. Site 17), producing abraided appearance. Flaring of the IIIS terminus wassuppressed in some nuclei of S. balcanicum (e.g. Sites10, 15, and 17), S. turgaicum (Site 17; Fig. 8A), andS. takahasii (Site 9).

More than 110 inversions were found among 480larvae, with 59 in S. balcanicum (N = 165 larvae), 41in S. lineatum (N = 88 larvae), six in S. takahasii(N = 49), and 14 in S. turgaicum (N = 178). Norearrangements were significantly linked (P > 0.05)to gender in any population. The sex chromosomes,therefore, were microscopically undifferentiated(X0Y0) in all populations. Additional rearrangements,particularly heterobands, were distributed among thefour species (Tables 2, 3).

SIMULIUM BALCANICUM

Ten sites had populations of S. balcanicum (N = 165).Two inversions, IS-4 and IL-11 (Fig. 5), were fixed inall individuals of S. balcanicum, and three additionalinversions (IL-14, IIIL-9, and IIIL-10) (Figs 4, 10A)were almost fixed (> 0.98), with only two larvaeheterozygous for IL-14 and one heterozygous forIIIL-9 and IIIL-10. Three additional inversions hadhigh representation in S. balcanicum across sites.IL-1 (Fig. 5B) was found in 61.8% of all homologues,and IIS-1 (Fig. 6) in 39.7%. The latter inversionserved as the base sequence on top of which at leasteight additional inversions were built (Table 2). IIL-1(Fig. 7) occurred in 91.2% of all homologues acrosssites, and was fixed in Turkish samples. InversionIIL-7 (Fig. 7), which occurred only on top of IIL-1,

Figure 2. Chromocentre of male larva of Simuliumturgaicum, showing bases of six chromosome arms (IS–IIIL), B chromosome (B), and nucleolar organizer (N.O.)



reversed the majority of the IIL-1 inversion backinto the standard orientation. Approximately 50additional polymorphic inversions and three rareheterobands (Figs 6, 9) were found across the sampledrange of S. balcanicum (Table 2). Five of six inver-sions with representation sufficient for analysisin three populations (Sites 10, 12, and 15) were inHardy–Weinberg equilibrium (Table 2).

Mean heterozygosity varied from 1.0 in Germanyto approximately 3.8 in Turkey, with one larva inTurkey having seven heterozygous inversions. Thir-teen polymorphisms were shared by half or more ofthe 10 populations. Within the hyperactive IIS arm,inversion breakpoints were clustered in threehotspots (sections 42–44, 45–46, and 50–53; Fig. 6).

Collection sites of S. balcanicum showed significant(P < 0.01) correlations between distance and degreesnorth (r = 0.942) and degrees east (r = 0.962). Regres-sion analysis indicated that distance between sites

followed a north-west to south-east axis; thus, sites innorthern locations were found west of sites in moresouthern locations (Fig. 12). The DDR analysis showeda significant (P < 0.01) negative relation between simi-larity of inversion profiles of S. balcanicum and dis-tance; as the distance between sites increased along anorth-west to south-east axis, the similarity of inver-sion frequencies between sites decreased (Fig. 13). Ofthe four inversions analyzed individually, two (IL-13and IIS-1) showed significant (P < 0.05) log-linear rela-tionships with direction (Table 4).

SIMULIUM LINEATUM

The six European populations of S. lineatum (N = 88)were united by high frequencies (> 0.15) of sharedinversions (IS-4, IS-6, IL-10, IL-11, IIL-8, IIL-9, IIL-11, and IIIL-5). Of 41 total inversions, 28 were foundin England (N = 49 larvae), 21 in Germany (N = 19),

Figure 3. Standard sequence for IS arm of female larva of Simulium turgaicum. C, centromere; N.O., nucleolarorganizer. Limits of inversions IS-1–IS-18 of various species are indicated by arrows or brackets. Ordering fragmentsindicated by the letters ‘a’ through ‘h’ will produce the IS-4, 6 sequence in Simulium lineatum.



11 in Italy (N = 6), nine in France (Corsica) (N = 12),and eight in the single larva from Poland. The meannumber of heterozygous inversions per larva rangedfrom 1.3 in Italy to more than 4.3 in England andGermany, with one larva in Germany having eightheterozygous inversions and two in England having13 each. Of the 11 inversions common to England andGermany, four [all in high frequency (> 0.50) inGermany] were fixed (IS-4, IL-10, and IIIL-5) or

almost fixed (0.98 for IL-11) in England. With fiveexceptions (IL-3b, IL-17, IL-18, IIL-10, and IIL-12),all inversions unique to either England or Germanywere in low (< 0.09) frequency. All inversions forwhich genotypic frequencies were adequate for statis-tical analysis (Site 5: IIL-8 and IIL-9; Site 18: IS-6,IL-3b, IL-17, IL-18, IIL-8, and IIL-11) were in Hardy–Weinberg equilibrium (Table 3). Six heterobands werefound (Figs 4, 7, 9; Table 3).

Figure 4. Standard sequence for IL arm of female larva of Simulium turgaicum. C, centromere; hb, location ofheteroband; m, marker (sensu Rothfels et al., 1978). Limits of inversions IL-2–IL-21 of various species are indicated bybrackets. The limits of inversion IL-14 of Simulium balcanicum are bracketed.

Table 4. Simple linear regression between the frequency of an inversion across sites (N = 8) for Simulium balcanicumand latitude and longitude; response and predictor variables were log10 transformed

Inversion Gradient Slope r2(adj) F1,6 P

IL-1 Latitude 3.67 0.104IL-1 Longitude 2.35 0.176IL-13 Latitude 0.0247 52.6% 8.76 0.025IL-13 Longitude 3.74 0.101IIS-1 Latitude –0.0321 73.1% 20.00 0.004IIS-1 Longitude 0.0198 78.6% 26.76 0.002IIL-1 Latitude 1.25 0.306IIL-1 Longitude 1.80 0.228



The English population demonstrated completeinversion linkage within chromosomes; the invertedsequences for IS-12, IS-13, IS-14, IS-15, and IS-16(Fig. 3) were traced on the same homologue (i.e. in cisconformation) in all seven larvae (six females, onemale). These same seven larvae also showed 100%linkage (cis conformation) of IIL-15 and IIL-16(Fig. 7). None of these IS or IIL inversions appearedindependently. We cannot be certain, however, thatthe IS and IIL inversions came from the same parent.The complement of these seven individuals was nomore loosely paired than that of typical individuals.

The following eight inversions in our study of S.lineatum were equivalent to those (in parentheses)found by Weber & Grunewald (1989): IS-4 (= IS-4),IS-5 (= IS-6), IIS-14 (= IIS-5), IIL-8 (= IIL-7), IIL-9

(= IIL-3), IIL-11 (= IIL-5), IIL-12 (= IIL-4), and IIL-14(= IIL-8).

SIMULIUM TAKAHASII

One sample of S. takahasii (N = 49) from Japan wasavailable for study (Table 1). All 49 larvae had thesame banding sequence, with two fixed inversions,IIIS-3 (Fig. 8B) and IIIL-10 (Fig. 10C). A darklystaining, glassy band of variable thickness washomozygously inserted in section 99 of all larvae;the chromosome typically was constricted at theband (Fig. 10C). Polymorphisms were minimal (fre-quencies < 0.02), with only four floating inversions,heterozygous expression of the nucleolar organizer,and a heterozygous band insert in section 45 of IIS

Figure 5. Chromosome I of female larva of Simulium balcanicum. A, IS (sections 4–9 only). The IS-4 sequence is present,with limits bracketed. Limits of inversions IS-6 and IS-9 are indicated by brackets. B, IL (sections 31–41 only). IL-1, IL-11,and IL-14 are present, with inversion limits indicated by brackets (IL-1, IL-14) or arrows (IL-11). Ordering fragmentsindicated by the letters ‘a’ through ‘h’ will produce the standard sequence. Limits of inversions IL-13, IL-15, and IL-16are indicated by brackets.



(Fig. 6, Table 3). The level of inversion heterozygo-sity in the population, consequently, was low(0.12).

SIMULIUM TURGAICUM

The three West Asian populations of S. turgaicum(N = 178) were characterized by a low level of poly-morphism. Of 14 total inversions, 13 were found incentral Turkey (N = 143 larvae) and one in Iran(N = 34), all with frequencies of 0.01 or less (Table 3).IIIS-1 in our system differed by one band from IIIS-1of Weber & Grunewald (1989). The mean number ofheterozygous inversions per larva was less than 0.15per site. Large supernumerary (B) chromosomes werepresent in one female and two male larvae fromTurkey. They paired at one end with the chromocentrein approximately 40% of the nuclei, suggesting anear-terminal centromere (Fig. 2). The puffed distalregion possibly signalled the presence of a nucleo-lar organizer (Procunier, 1975). Two heterobandsin low frequency (< 0.01) were discovered (Figs 4, 8;Table 3).

CHROMOSOMAL COMPARISONS

The distribution of inversions differed significantlyamong the four species (χ2 = 47.06, d.f. = 4, P < 0.001)(Table 5). Simulium balcanicum differed from allother species by two fixed inversions (IS-4 andIL-11), two almost fixed (i.e. frequencies ≥ 0.98)inversions (IL-14 and IIIL-9), and a unique set ofpolymorphisms. It shared IIIL-10 with S. takahasii,which had one unique fixed inversion (IIIS-3). Norearrangements were shared between S. balcanicumand S. turgaicum. However, five inversions wereshared between S. balcanicum and S. lineatum: IS-4,IL-11, IIL-8, IIL-11, and IIL-12. The number of inver-sions across chromosome arms was not independentof the two species (χ2 = 29.894, d.f. = 4, P < 0.001; IIISwas omitted from analysis because of only one inver-sion) (Table 5). Simulium lineatum, S. takahasii, andS. turgaicum shared no rearrangements. Simuliumlineatum could be distinguished from S. turgaicum byits high frequency of inversions (e.g. IS-4, IL-10,IL-11, IIL-9, and IIIL-5), which typically were presentin half or more of the chromosomal homologues ateach site. No hybrids were detected at any sites where

Figure 6. Standard sequence for IIS arm of female larva of Simulium turgaicum. Limits of inversions IIS-1–IIS-32 areindicated by arrows or brackets. Inversions above the chromosome are specific to Simulium balcanicum; those below thechromosome are specific to Simulium lineatum. Inversions IIS-2, IIS-3, IIS-9, IIS-10, IIS-20, IIS-26, and IIS-31 occur onlyon top of the IIS-1 inversion. Bu, bulge; C, centromere; hb, location of heteroband; i, location of band insert; RB, ring ofBalbiani; ss, shoestring marker; the two halves of the bulge are separated by the ring of Balbiani. Ordering fragmentsindicated by the letters ‘a’ through ‘h’ will produce the IIS-1, 2 sequence of S. balcanicum.



S. balcanicum occurred in the same rivers withS. lineatum (Germany) and S. turgaicum (Turkey).

MORPHOLOGICAL COMPARISONS

Only the gill of S. balcanicum, with its petiolatepair of filaments, was consistently distinguishablefrom the gills of the other three species. A principal

components analysis of gill lengths and selected ratiossuggested that S. takahasii was distinct but thatS. turgaicum was indistinguishable from S. lineatum,although sample sizes were small (Fig. 14). The firsttwo axes of the principal components analysisexplained 83.7% of the total variation in gill morphol-ogy; therefore, only these two were used to graph gilldimensions in multidimensional space.

Figure 7. Standard sequence for IIL arm of female larva of Simulium turgaicum. C, centromere; DNA, DNA puff; dt,doublet; gB, grey band; hb, location of heteroband; j, jagged marker; Pb, parabalbiani; pf, puffing band. Limits ofinversions IIL-1–IIL-21 of various species are indicated by brackets. Ordering fragments indicated by the letters ‘a’through ‘h’ will produce the IIL-9, 14 sequence of Simulium lineatum, and ordering the letters ‘a’ through ‘f ’ will producethe IIL-1, 7 sequence of Simulium balcanicum.

Table 5. Distribution of all inversions, fixed and floating, in each chromosome arm of 4 species in the subgenus Wilhelmiaacross 18 sites

Larvae (N) Inversions (N) IS IL IIS IIL IIIS IIIL

Simulium balcanicum 165 59 5 6 31 8 0 9Simulium lineatum 88 41 13 8 2 13 1 4Simulium takahasii 49 6 0 1 0 2 2 1Simulium turgaicum 178 14 1 8 0 1 1 3



DISCUSSION

Our samples can be partitioned into four chromo-somal segregates, which we recognize as sepa-rate species (S. balcanicum, S. lineatum, S. taka-hasii, and S. turgaicum), each supported byunique cytogenetic characters and geographicaldistributions.

These four species can be distinguishedchromosomally at a glance from two sympatricmembers of the subgenus Wilhelmia, S. equinumand S. pseudequinum Séguy, by the presence ofa chromocentre (Weber & Grunewald, 1989;Chubareva & Petrova, 2008). Although ectopicpairing of centromeres occurs in some populations ofS. equinum, it is not present in all nuclei, and theexpanded centromere region of chromosome I inS. equinum contrasts markedly with the slender con-dition in S. balcanicum, S. lineatum, S. takahasii,and S. turgaicum. The positions of the bulge andring of Balbiani also are diagnostic. In our four taxa,they are in the distal one-third of the arm whenin the standard sequence, with the bulge proximalto the ring of Balbiani, whereas, in S. equinumand S. pseudequinum, they are subterminal, withthe bulge distal, unless displaced by floating inver-sions (Weber & Grunewald, 1989). Simuliumparaequinum, which also has a chromocentre, hasthe bulge and ring of Balbiani in the proximal one-third of the arm, nearer the centromere (Petrovaet al., 2003).

SPECIES STATUS

Our material of S. balcanicum from Germany andTurkey almost brackets the geographical range, whichextends from Italy (Rivosecchi, 1978) and our collec-tion site in Germany eastward to the Krasnodar Ter-ritory of Russia (Grinchuk & Chubareva, 1974). Oursample from Bârza, Romania, is within 200 km of thetype locality (Lakatnik) in the Balkan Mountains ofBulgaria. The chromosomes reveal unequivocally thatS. balcanicum is a distinct species, with no evidence ofcryptic species, across its range of approximately2000 km. Polymorphisms unique to one or a few popu-lations are in low frequency, typically less than 0.10per site. Common inversions exhibit a longitudinalgradient of frequencies, with a north–south tilt,although whether the frequencies are driven by selec-tion in specific environments or by restricted gene flow,or both, is unknown. Inversion-frequency gradientsare common in a number of simuliid species, typicallygrading toward loss or fixation at the distributionalperiphery (Rothfels & Featherston, 1981), a patternalso documented in Drosophila (Stocker, Foley &Hoffmann, 2004). We conclude that our samples ofS. balcanicum are conspecific with the type specimenand with the types representing the three synonyms(secundum, danubiense, and severinense). The petio-late pair of pupal gills, originally described by Rubtsov(1956), provides the only diagnostic structural featurefor S. balcanicum in any life stage (Crosskey & Zwick,2007).

Figure 8. Chromosome IIIS of Simulium (Wilhelmia) species. bl, blister; C, centromere; ca, capsule; hb, heteroband(shown as heterozygous). A, standard sequence for IIIS arm of female larva of Simulium turgaicum, showing suppressedterminal flaring (sections 72–73). Limits of inversions IIIS-1 and IIIS-2 and fixed inversion IIIS-3 are indicated bybrackets; autosomal inversion IIIS-4 of Simulium takahasii, indicated by arrows, occurs only on top of IIIS-3; the twoinversions share near-coincident proximal breakpoints such that IIIS-4 is based on the fixed IIIS-3 sequence. B, malelarva of S. takahasii; IIIS-3 and IIIS-4 as a heterozygote (inverted on lower homologue) are present.



As S. balcanicum increases in prevalence eastward,S. lineatum diminishes in prevalence and is replacedby S. turgaicum. Where S. balcanicum overlaps geo-graphically with S. lineatum, it typically occurs indifferent rivers. Our additional, albeit limited, find-ings of S. balcanicum in south-eastern Germany (G.Seitz, unpubl. data) suggest that it might be expand-ing its range westward.

Our German sample of S. lineatum probably repre-sents the type specimen of S. lineatum; the type local-ity in Stolberg, Germany, is approximately 490 kmnorth-east of our nearest sampling site (Landshut).Our English sample of S. lineatum is approximately215 km south of the type locality of S. salopiense andapproximately 1000 km west of our German sample.English and German populations differ only in thefrequency of rearrangements and the presence ofunique, rare inversions, which are not unexpected,

given the geographical distance. We, therefore, agreewith the synonymy of salopiense with lineatum origi-nally proposed by Crosskey & Davies (1972).Although similarly having a high frequency (> 0.15) ofthe same inversions (IS-4, IS-6, IL-10, IL-11, IIL-8,IIL-9, IIL-11, and IIIL-5) as in England and Germany,the Corsica and Italian populations have fewerpolymorphisms, perhaps reflecting their locations atthe periphery of the distribution.

Our analyses provide no concrete evidence forcryptic species in S. lineatum s.s., nor did the analysisby Weber & Grunewald (1989) of populations insouth-western Germany. Thus, the currently recog-nized synonymy (Adler & Crosskey, 2014) of falcula(Enderlein) with lineatum is valid. One caveat,however, is in order. Our English population ofS. lineatum has the highest number of heterozygousinversions (N = 13) and of linked inversions (≥ 5) ever

Figure 9. Standard sequence for IIIL arm of female larva of Simulium turgaicum. C, centromere; hb, location ofheteroband; 2 + 1, marker with two black bands and one grey band, possibly homologous with bands in section 93 of thestandard sequence for the subgenus Simulium. Limits of inversions IIIL-1–IIIL-6, IIIL-8, and IIIL-11–IIIL-15 areindicated by brackets. Positions of heterobands in section 97 of Simulium balcanicum and one each in section 100 ofS. balcanicum (hb1) and Simulium lineatum (hb2), and of a band insert in section 99 of Simulium takahasii (i) areindicated by arrows. Ordering fragments indicated by the letters ‘a’ through ‘h’ will produce the IIIL-9, 10 sequence ofS. balcanicum.



recorded for a single simuliid larva (Adler et al.,2010). The seven individuals with linked inversions inIS and in IIL might represent products of introgres-sion, although we found no evidence of loose somatic

pairing of homologues typical of hybrid individuals(Rothfels & Nambiar, 1981). The only other knownspecies of the subgenus Wilhelmia in Britain areS. equinum and S. pseudequinum, and their chromo-somal features are markedly different from thoseof S. lineatum. The possibility of cryptic taxa inS. lineatum will require additional sampling, particu-larly in England.

We resurrect the name turgaicum from synonymywith lineatum and apply it to our West Asianpopulations (Armenia, Iran, and Turkey) formerlyknown as S. lineatum. Rubtsov (1956) describedS. turgaicum from Almaty, Kazakhstan, approxi-mately 2500 km east of our nearest sampling site(Iran), and reported that the larvae and pupae occur insemidesert, small, warm rivers and irrigation chan-nels, as well as cold rivers up to elevations of 2000 m inthe Transcaucasus and Central Asia. Former Sovietstudies (Grinchuk & Chubareva, 1974) consistentlyidentified Armenian populations as S. turgaicum.

Figure 10. Chromosome IIIL of female larvae of Simulium (Wilhelmia) species. A, Simulium balcanicum (sections 83–92only) with the typical IIIL-9, 10 sequence; arrows indicate inversion breakpoints. Letters ‘a’ through ‘h’ indicate the sequenceof chromosome fragments necessary to produce the standard IIIL sequence from the IIIL-9, 10 sequence. C = centromere,1 + 2 = marker with one grey band and two black bands. Limits of inversion IIIL-7 are indicated by bracket. B, Simuliumturgaicum, showing the standard sequence (sections 89–94 only); arrow in section 92 indicates sticky heterochromatin; 2 + 1indicates the same marker as in section 89 of Fig. 9A but with reversed polarity. C, Simulium takahasii [sections 83–91(half)and 98–100 only] with fixed inversion IIIL-10 and characteristic band insert in section 99.

Figure 11. Pupal gill of Simulium lineatum from Corsica,France, showing numbering system for the eight filamentsand examples of measurements (lines) for filaments 1, 2,and 3.



The chromosomal distinction between S. lineatumand S. turgaicum cannot be evaluated in sympatry.Our nearest samples are approximately 2100 kmapart. Whether the distributions actually overlap isnot known. If S. lineatum and S. turgaicum aresympatric, the zone of overlap probably liesin southern Russia or in the Balkans, perhapsBulgaria, the easternmost reliable record forS. lineatum, based on the listing by Kovatschev(1979). One of the few putatively diagnostic featuresof S. turgaicum, the greater length of theanteriormost filaments of the pupal gill (Rubtsov,

1956), does not hold consistently. Some ecologicaldistinction, nonetheless, might exist. Simuliumlineatum is a lowland species, typically occupyinghabitats below 500 m a.s.l, whereas S. turgaicumis common at higher elevations (> 900 m). Thealtitudinal differences are reminiscent of siblingspecies in the S. ochraceum complex (Hirai et al.,1994). If the uniquely shared polymorphismsof S. balcanicum and S. lineatum are the result ofcommon ancestry, rather than introgression, thespecies status of S. turgaicum is further supportedby its relationship as sister to S. balcanicum plus

Figure 12. Reduced major-axis regression between latitude and longitude coordinates for eight sites from whichchromosomal inversion profiles of Simulium balcanicum were analyzed.

Figure 13. Distance–decay relationship between site similarity, based on chromosomal inversion profiles of Simuliumbalcanicum, and distance between sites.



S. lineatum, rather than to S. lineatum with whichit historically has been in synonymy.

The full geographical range of S. turgaicum isunknown but is considered here to extend from Turkeyeastward at least to the type locality in Kazakhstan. Itprobably extends well beyond Kazakhstan deep intoCentral Asia. Whether its range overlaps that ofS. takahasii is unknown. Simulium takahasii occupiesthe eastern perimeter of the Palearctic Region in east-ern China, Japan, and Korea (Adler & Crosskey, 2014);we consider the record of S. lineatum from Beijing(Adler & Crosskey, 2014) to apply to S. takahasii.

We continue to recognize S. takahasii as a validspecies, primarily on pragmatic grounds (to maintainthe current nomenclatural status) rather than onthe basis of the few chromosomal differences fromS. turgaicum. Whether S. takahasii is a valid speciesor conspecific with S. turgaicum suffers from thelack of an opportunity to test reproductive isolationin sympatry. Our collections of S. takahasii andS. turgaicum are separated by more than 7250 km.This geographical gap leaves open the question ofwhether some or all of the additional, putative speciesof S. (Wilhelmia), described from China and basedon questionable structural characters, might beconspecific with S. turgaicum or S. takahasii, includ-ing Simulium germuense Liu, Gong, Zhang, Luo &An; Simulium pekingense Sun; Simulium qinghaienseLiu, Gong, Zhang, Luo & An; Simuliumqingxilingense Cai & An; Simulium tachengenseMahe, An & Yan; Simulium tongbaishanense Chen &Luo; and Simulium xingyiense Chen & Zhang. Only

the polytenes of S. xingyiense have been published(Huang et al., 2012), although the low-magnificationresolution of the maps is not sufficient to allow com-parison with our populations.

CHROMOSOMAL RELATIONSHIPS

Our evaluation of the relationships of the four taxa isinfluenced by the choice of the standard bandingsequence and by the scrambled sequence of the com-plement, which currently does not invite comparisonwith sequences of known taxa that could serve asoutgroups. We can determine the polarity of onlyone relevant band sequence through outgroup com-parison. Our standard sequence for IL-14 (sections40–41) is equivalent to the standard sequence forthe Simulium vernum group (sections 42C–44C,Brockhouse, 1985), the subgenus Simulium (sections39c–41, Rothfels et al., 1978), and Simuliumerythrocephalum (De Geer) (P. H. Adler, unpubl.data). If these taxa are taken as outgroups for thissequence, IL-14 of S. balcanicum is derived.

The chromosomes indicate that S. balcanicumand S. lineatum are more closely related to oneanother than either is to S. turgaicum. Although therelationships are not rooted, the same autosomalpolymorphisms (e.g. IIL-8, IIL-11, and IIL-12) areuniquely shared regardless of the chromosomalsequence chosen as the standard; thus, the sisterrelationship of S. balcanicum and S. lineatum holds.The caveat, however, is that we do not know whetherthe shared rearrangements are primary (derived from

Figure 14. Principal components (PC) analysis of the lengths of all gill filaments and ratios of filaments 5 : 2 and 3 : 1of four species of Simulium (Wilhelmia). The first PC is largely a linear measure of filaments, and the second PC is largelya measure of proportions.



a common ancestor) or secondary (acquired by intro-gression). Simulium balcanicum and S. lineatum arechromosomally most similar in the area of overlap(Germany), where they share five inversions.

On the basis of one uniquely shared inversion(IIIL-10), S. takahasii is most closely related toS. balcanicum. The relationship, however, might bean artefact of our choice of standard. In other words,if IIIL-10 is actually ancestral, our standard sequencefor this inversion becomes the derived condition andthe relationship of S. takahasii with S. balcanicum isnot supported.

PEST PROBLEMS

The attacks on livestock and humans along Turkey’sKizilirmak River (Yilmaz et al., 2007; Sariözkan et al.,2014) are the most severe recorded for any member ofthe subgenus Wilhelmia. Citizens in Isfahan, Iran,where we have cytotyped S. turgaicum, have com-plained about biting insects, although the situation isobfuscated by a failure to distinguish black flies fromthe common Culex mosquitoes in the area. Simuliumlineatum is a pest of livestock and occasionally ofhumans in parts of Europe (Crosskey, 1990; Werner &Adler, 2005), and S. takahasii is a pest of cattle andhorses in Japan (Bentinck, 1955). No pest problemshave been reported for S. balcanicum, although theabsence of reports might be related to the inability todistinguish the isomorphic females of this speciesfrom those of S. lineatum and S. turgaicum. Peststatus of the group members is related to theirshared propensity to feed on large mammals and toproduce large populations in productive rivers andirrigation ditches near human and livestock foci. TheCappadocia outbreak probably represents an extremeexample of these factors.

GEOGRAPHICAL GENERALISTS AND SPECIALISTS

The existence of four discrete species, rather thanone, reinforces a general pattern in the Simuliidae:wide distributions of putatively single species repre-sent mosaics of geographically restricted crypticspecies. This is not to say that all species of black flieswith large geographical distributions are species com-plexes. Legitimate species with expansive ranges canreflect the ability to capitalize on anthropogenicallyaltered environments (Adler & Kim, 1984). The trend,nonetheless, offers a guide for screening putativespecies for additional biodiversity. Accordingly,S. turgaicum, with the largest range of our four sur-veyed taxa and which has been minimally sampled,might consist of yet additional species.

The discovery that widespread black flies are com-posites of geographical specialists largely reflects the

opportunity provided by the well-banded polytenechromosomes for whole-genome inspection of naturalpopulations. The same trend is apparent in othergroups of organisms that offer the polytene option,notably mosquitoes and drosophilids (Coluzzi et al.,2002; Rohde et al., 2006). This phenomenon, however,is not unique to black flies and other flies with high-quality polytenes. Variation in life-history aspectssuch as habitats, hosts, phenologies, mating behav-iours, and communication systems have long sig-nalled complexes of cryptic species (Mayr, 1963;Bickford et al., 2006). As genomic analyses becomemore powerful and routine, the opportunity fortesting hypotheses of generalist species versus com-plexes of specialists will become more democratized.

ACKNOWLEDGEMENTS

We thank C. E. Beard for excellent technical assis-tance and C. L. Brockhouse and three anonymousreviewers for their insightful comments on the manu-script. This work was funded, in part, by NationalScience Foundation awards DEB-0841636 (Discoveryand Prediction of Hidden Biodiversity in Black Flies(Diptera: Simuliidae)) under the American Recoveryand Reinvestment Act of 2009 and DEB-0933218(MIDGEPEET: A Collaborative Effort to IncreaseTaxonomic Expertise in Understudied Families ofNematocerous Diptera). Funding also came fromThe Scientific and Technological Research Council ofTurkey Research Project 111 O 426 ‘The MolecularClassification of Black Fly (Diptera: Simuliidae)Species Which Pose a Problem in Central KizilirmakBasin and the Investigation of Their Vector Potentialsby Real Time PCR’, VEGA grant no. 1/0561/14 of theMinistry of Education of the Slovak Republic, and theSlovak Research and Development Agency under con-tract No. APVV-0436-12. This is Technical Contribu-tion no. 6253 of the Clemson University ExperimentStation, and is based on work supported in part byNIFA/USDA under project number SC-1700433.

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