Population variability in Chironomus (Camptochironomus) species (Diptera, Nematocera) with a Holarctic distribution: evidence of mitochondrial gene flow

Insect Molecular Biology (2002)

11

(5), 387–397

© 2002 The Royal Entomological Society

387

Blackwell Science, Ltd

Population variability in

Chironomus

(

Camptochironomus

) species (Diptera, Nematocera) with a Holarctic distribution: evidence of mitochondrial gene flow

J. Martin*, V. Guryev† and A. Blinov†

*

Genetics Department, University of Melbourne, Victoria, Australia;

†

Laboratory of Cell Biology, Institute for Cytology and Genetics, Novosibirsk, Russia

Abstract

Phylogenetic analysis of DNA sequences from mito-chondrial (mt) genes (

Cytochrome b

and

Cytochromeoxidase I

) and one nuclear gene (

globin 2b

) was usedfor the investigation of Nearctic and Palearctic popu-lations representing four

Chironomus

species of thesubgenus

Camptochironomus

, namely

C. biwaprimus

,

C. pallidivittatus

,

C. tentans

sensu stricto and

C. dilutus

(the last two species constitute Holarctic

C. tentans

sensu lato). Phenograms constructed on the basisof mt sequences were not congruent with treesbased on nuclear genes, or with morphological andcytological data. The mt tree divided the populationsby continental region, rather than by the speciesgroupings recognized by the other data sets. Theincongruence is explained by mt gene flow resultingfrom hybridization between the sympatric species oneach continent. Calculation of divergence times, basedon the sequence data, suggest that

C. tentans

(s.l.) and

C. pallidivittatus

have both been in North America forabout 2.5 My.

Keywords:

Chironomus

, mtDNA, globin, mitochondrialgene flow, molecular phylogeny, Holarctic.

Introduction

In insects, most supposedly Holarctic species have beenrecognized on purely morphological criteria withoutsupporting studies of inter- and intrapopulation genetic vari-

ability. Consequently there are few data to indicate the extentof intraspecific differentiation within and between conti-nents, and where such data do exist they may be subject todiffering interpretations (e.g. Tauber & Tauber, 1987). TheChironomidae are an ideal group in which to investigatedifferentiation and migration of Holarctic species. Theypredominate the Arctic fauna (Downes, 1962) and it has beenestimated that 25–33% of all Nearctic or Palearctic chiro-nomid species are truly Holarctic (Cranston & Oliver, 1987).The Arctic and Boreal species are assumed to have beeninitially Palearctic and to have spread to North America(NA) during the Pleistocene. Many species possess poly-tene chromosomes, which permit accurate determinationof variability based on the level of fixed and polymorphicchromosomal rearrangements between populations(Martin, 1979) to assess whether Nearctic and Palearcticpopulations do constitute a single Holarctic species.

Recent cytogenetic studies of Holarctic

Chironomus

spe-cies (Kiknadze

et al

., 1996, 1998, 2000; Butler

et al

., 1999) haverevealed markedly different levels of differentiation betweenNearctic and Palearctic populations in different species. Somespecies, such as

C. thummi

, have essentially the samebanding pattern of the polytene chromosomes on bothcontinents (J. Martin, unpublished list of North American

Chironomus

-species, http://www.genetics.unimelb.edu.au/Martin2/NACytfiles/NAChiron.html). Others, such as thepopulations of

C. tentans

, have very different bandingpatterns in the two continents (Kiknadze

et al

., 1996), thedifferences being sufficiently great that the name

C. dilutus

was proposed for the Nearctic populations (Shobanov

et al

.,1999). In addition, the Nearctic populations show significantcytological differentiation, such that Acton & Scudder(1971) recognized three races: Alaskan, West Canadian(WC) and East Canadian (EC) races. While the latter twoshould be recognized as

C. dilutus

, the status of theAlaskan race is uncertain because it has either beenplaced as a more recent derivative of

C. tentans

(Acton &Scudder, 1971) or intermediate between Palearctic andNearctic populations (Kiknadze

et al

., 1996). The introduc-tion of a separate name for the Nearctic populationsrequires a distinction when referring to the Holarctic entity

Received 14 January 2002; accepted after revision 30 April 2002. Corres-pondence: Dr Jon Martin, Genetics Department, University of Melbourne,Victoria 3010, Australia. Tel.: 613 83446258; fax: 613 83445139; e-mail:[email protected]

IMB_348.fm Page 387 Wednesday, August 28, 2002 10:36 AM

http://www.genetics.unimelb.edu.au

388

J. Martin, V. Guryev and A. Blinov

© 2002 The Royal Entomological Society,

Insect Molecular Biology

,

11

, 387–397

[

C. tentans

sensu lato (s.l.)] and to the Palearctic com-ponent [

C. tentans

sensu stricto (s.s.)].

C. pallidivittatus

, another sibling species of

C. tentans

(s.l.), has differentiated to a much lesser extent in the twocontinents (Kiknadze

et al

., 1998). There is, however, noevidence whether such differences in the level of differenti-ation could be attributed to migration at different timesor to different rates of cytological evolution in the variousspecies. The greater level of cytological variation observedin European populations of both

C. tentans

(s.l.) and

C. pallidivittatus

, together with the evolutionary relation-ships of the chromosome banding patterns, suggest thatthe Palearctic populations of these species are ancestralto those of the Nearctic. The presence of a chromosomebanding sequence in the most easterly Siberian sample of

C. tentans

(s.s.), which was otherwise found only in NApopulations, led Kiknadze

et al

. (1996) to conclude thatmigration into North America had occurred by way of theBering bridge and Alaska.

These latter sibling species belong to the subgenus

Camptochironomus

, which also includes another siblingspecies,

C. biwaprimus

that is endemic to Japan, andhas been described on morphological grounds as closeto

C. tentans

(s.l.) (Sasa & Kawai, 1987). These speciesform a discrete cluster (Cluster III) in Guryev

et al

.’s

(2001) molecular phylogeny of

Chironomus

. The aim ofthe present study is to compare the molecular data withpublished data obtained from cytological analysis (Kiknadze

et al

., 1996, 1998) to reconstruct the evolutionary historyof the Holarctic sibling species. Also we will assess thelevel of inter- and intracontinental molecular differentiationwithin

C. tentans

(s.l.) and

C. pallidivittatus

and estimateseparation times to address the possible distributionpattern of these species and the evolutionary positionof

C. biwaprimus.

Both nuclear and mitochondrial (mt) genes have beenused because current evidence indicates that the multipleindependent genes enhance the reliability of phylogeneticinference and enable gene tree effects to be recognized(Cummings

et al

., 1995; Maddison, 1997).

Results

We sampled twelve populations of

C. tentans

(s.l.) (includ-ing five of

C. dilutus

), eight populations of

C. pallidivittatus

from the Palearctic and Nearctic, and one population of

C. biwaprimus

from Japan (Table 1). Only the

gb2b

sequences were available for four samples. Thesewere the German populations of

C. tentans

(s.s.) and

C. pallidivittatus

, which came from previously published

Table 1. Populations sampled and their geographic origin

Location Abbreviation Cytb COI gb2b

C. tentans s.s.Yakutia, East Siberia, Russia Yak + + +Altai, West Siberia, Russia Alt + + +Semipalatinsk, Kazakhstan Semi + + +Stockholm, Sweden Stoc Laboratory population; Karolinska Institute, Stockholm, Sweden + + +Lund, Sweden Lund Laboratory population; University of Lund, Sweden + + +Germany Germ Hankeln et al., 1997 – – +Anchorage, Alaska, USA AAK Alaskan Race – – +

C. dilutusFuller’s Slough, ND, USA FND West Canadian Race + + +Hankenson, ND, USA HND West Canadian Race + + +Lake Christina, MN, USA CMN West Canadian Race + + +Winnipeg, Man, Canada WCA Laboratory population; University of Melbourne, Australia; West Canadian Race + + +Longmeadow, MA, USA LMA East Canadian Race + + +

C. pallidivittatusYakutia, East Siberia, Russia Yak + + +Altai, West Siberia, Russia Alt + + –Omsk, West Siberia Omsk + + +Lund, Sweden Lund Laboratory population; University of Lund, Sweden + + +Germany Germ Hankeln et al., 1997 – – +Fuller’s Slough, ND, USA FND + + +Cleveland, ND, USA CND + + +Jamestown, ND, USA JND – – +

C. biwaprimusLake Biwa, Honshu, Japan + + +

C. thummiSaratov, Russia Laboratory population: Institute of Cytology and Genetics, Novosibirsk, Russia + + +

C. oppositus f. connoriBellerive, Tas., Australia Martin and Lee, 1984 + + +


Mitochondrial gene flow in

Camptochironomus 389



,

11

, 387–397

data (Hankeln

et al

., 1997), as well as the Alaskan popula-tion of

C. tentans

and the Jamestown ND population of

C. pallidivittatus

, both of which had been fixed in aceticethanol for long periods.

Phylogenetic relationships inferred from

gb2b

and mt sequences

Nucleotide (nt) sequences obtained from the fragment ofthe

gb2b

gene can be divided into two groups: (i) samplesof

C. tentans

(s.l.) and (ii) samples of

C. pallidivittatus

and

C. biwaprimus

(Table 2). Six of the twelve nt differencesthat clearly distinguish these two groups lead to changesof amino acid residues. In general the corrected nt differ-ences between samples of the same species were lessthan 2%, with two exceptions: (i)

C. tentans

(AAK), inwhich a basal position is suggested by it being closestto the

C. pallidivittatus

/

C. biwaprimus

cluster; and (ii)

C. pallidivittatus

(JND), which lacks some of the substitu-tions by which all the other

C. pallidivittatus

samples differfrom the

C. tentans

(s.l.) (Tables 2 and 3).Tamura-Nei (TN) distances of the nt sequences were

calculated separately for

Cytb

and

COI

and were highly con-gruent, as found by Guryev

et al

. (2001), whose data includedseveral of the sequences used here. The data were thereforeconcatenated for subsequent analysis, for the reason givenby Guryev

et al

. (2001). The corrected nt sequence diver-gence between samples within species was generally lessthan 2%, similar to those for the

gb2b

comparisons,although between NA samples from the West and EastCanadian races of

C. dilutus

the divergence ranged from2.1% to 3.6% (Table 3). In contrast, the divergence betweenthese samples and those of NA

C. pallidivittatus

wasonly 1.1–2.9% and between Palearctic

C. pallidivittatus

and

C. tentans

(s.s.) only 1.6–2.4%. Divergence betweenpopulations of different species on different continentswas greater, at about 5.6–8.9%, similar to that between

C. biwaprimus

and each of the other species.The inferred phylogenetic tree based on

gb2b

sequences is shown in Fig. 1. Exclusion of the four samplesfor which mitochondrial genes were not studied did not alterthe topological relationships of the other populations (datanot shown).

Not surprisingly, the populations were divided into twoclusters, as indicated by the analysis of nt sequences.There is strong bootstrap support for the monophyleticnature of the

C. tentans

(s.l.) cluster, but rather lessbootstrap support for

C. pallidivittatus

/

C. biwaprimus

asa monophyletic group. In the

C. tentans

(s.l.) group theAlaskan sample (AAK) is basal to all other populations. ThePalearctic and the Nearctic populations of

C. tentans

(s.l.)constitute two separate branches on the phylogenetic tree,with the exception that the East Siberian (Yak) populationof

C. tentans

(s.s.) is basal to the ‘Nearctic’ (

C. dilutus

)cluster. The terminal position of the latter suggests that NA

populations have been derived from Palearctic, possiblySiberian, populations

.

For

C. pallidivittatus

, a Nearctic/Palearctic separation isnot as strong as that for

C. tentans

(s.l.). The Europeansamples (Lund, Germ) cluster together (BP = 96/91/84),while the East Siberian (Yak) and West Siberian (Omsk)samples possibly constitute a group with the North Ameri-can ones (BP = 68/57/53). Topology of the tree suggeststhat

C. pallidivittatus

(JND) is basal for this cluster, althoughthis is not supported by a high bootstrap value.

Phylogenetic analysis, using the combined sequencedata for the two mt genes, produced the tree topologyshown in Fig. 2. The populations are again divided into twogroups but, in contrast to the

gb2b

gene analysis, thepopulations are grouped by geographical distribution, ratherthan according to the species to which they belong. Thistree supports two mixed clusters with Nearctic populationsof both species in the first, and all Palearctic populations inthe second cluster. In the Nearctic branch

C. dilutus

and

C. pallidivittatus

are intermixed, with only the grouping ofthe two

C. pallidivittatus

populations being supported bymoderately high BP values (78/84/85). On the other hand,the

C. tentans

(s.s.) and

C. pallidivittatus

populationsare relatively well separated from each other within the‘Palearctic’ branch, with

C. biwaprimus

the most divergentspecies within this branch.

Tests were performed using

PAUP

* (Swofford, 1998) toconfirm that the trees produced by the nuclear and mtsequences were significantly different. For the MP trees,Templeton’s non-parametric test (

n

= 21,

z

=

−

4.25) gave aprobability of less than 0.001 that there was no differencebetween the two trees. For the ML trees, the SH test (–lnL = 64.11) gave a probability at least as low, that there wasno difference between the tree topologies. We can there-fore conclude that the difference between the trees inFigs 1 and 2 reflect differences between the data sets, andnot random variations.

The relationships between the various populations andspecies can be seen in a useful way as the unrootedhaplotype network shown in Fig. 3. The haplotype networkfor the mt sequences is more complex than that for the

gb2b

sequences because some haplotypes showed multipleequally parsimonious connections to other haplotypes(Fig. 3B). This was most noticeable for the haplotypes ofNA

C. dilutus

, but also occurred with the Palearctic

C. pallidivittatus

haplotypes. The networks, calculatedseparately for

Cytb

and

COI

, showed some differences fromeach other, mainly with respect to the relationship of the

C. biwaprimus

sequence: in the case of the

COI

network,the

C. biwaprimus

haplotype was derived from those of

C. pallidivittatus

, as in the

gb2b

network (Fig. 3A). Theseparate mt networks were also similar to the

gb2b

networkin that the

C. biwaprimus

haplotype was less removed fromthe other Palearctic haplotypes than they were from the NA


390

J. Martin, V. Guryev and A. Blinov



,

11

, 387–397

Table 2. Polymorphic sites in the sequence alignments obtained in this study. Asterisks under gb2b alignment represent sites with amino acid replacement which differ between C. tentans (s.l.) and C. pallidivittatus

gb2b 1111111111111222222 33345666001456667888900337936689243562852301653896237273

C. tentans Yak CCCTTCCAACCCCGAGAAGTGCGAATCGCC. tentans Alt ................TC..A........C. tentans Semi ................TC..A........C. tentans Stoc ................TC..A......C.C. tentans Lund ................TC..A........C. tentans Germ .........A......TC..A........C. tentans AAK ??.C..T.....G....C...........C. dilutus FND ...........T.T...C...........C. dilutus HND ...........T.....C...........C. dilutus CMN ..T..........T...C...........C. dilutus WCA .............T...C...........C. dilutus LMA .............T...C...........C. pallidivittatus Yak ?T.CCAGGG.T.G..ACCC.AT.GG....C. pallidivittatus Omsk ?T.C.AGGG.T.G..ACCC.AT.GG....C. pallidivittatus Lund .T.C.AGGG.T.G.TA.C.CA..GG....C. pallidivittatus Germ .T.C.AGGG.T.G..A.C.CA..GG...TC. pallidivittatus FND ?T.C.AGGG.T.G...TCC.AT.GG....C. pallidivittatus CND TT.C.AGGG.T.G..ATCC.AT.GG....C. pallidivittatus JND ?T.C.ATG....G..ATC..AT.GG....C. biwaprimus .T.C.AGGG...G..ATC..ATAGGCT..

* * * * **

COI 1111111222222222223333333333333333333444444444445555555555555566 4556677835566990122234458801122334467777888990566778899902345557888999011958173650146909210698144067365847367016725818610608470192758039736925810

C. tentans Yak ACCTATCCCAACAATAAATTTTATTAACCTTTCCTCCTTCCTAATTATTACTATTAATTACTCTTCCGAGAATC. tentans Alt T...........................T..................................C.T.A.....C. tentans Semi ????..........?????????????.T..................................C.T.A.....C. tentans Stoc ......................G.....T.C....................................A.....C. tentans Lund ......................G.....T.C....................................A.....C. dilutus FND CTTC..TT..GT..CGC....ACCC..T...C..G.TCCTT..G....C.T.GC.G......TCA.TA.AGG.C. dilutus HND ?TTC..TT..GT..CGC....ACCC..T...C..G.TCCTT..G....C.T.GC.G......TCA.TA.AGG.C. dilutus CMN .TTCT.TT..GT..CGC...C.CCC..T...C..G..CCTT..G.C..C.TCGC.T..CT.CTCA.TA.AGGCC. dilutus WCA ?TT...TT..GT..CGC....ACCC..T...C..G..CCTT..G....C...GC.G...G..T.A.TA.AGG.C. dilutus LMA ?AT.CCTT...T...GC....ACCC.GT...C....TCCTT..G..G.CGT..C........TCG.TAG..G.C. pallidivittatus Yak ................G.........G..C.....T............C..................A.A...C. pallidivittatus Alt ..............C.G.........G........T........C...C...........T.T....A.A...C. pallidivittatus Omsk ................G.....G.CGG........T............C.............TC...A.A...C. pallidivittatus Lund ................G.....G...G..C.....T............C.............T..T.A.A...C. pallidivittatus FND .TTC..TT..GT..CGC....ACCC.GT...CTTG.TCCTT..G....C.T..C.G......TCA.TA.AGG.C. pallidivittatus CND .TTC..TTT.GTG.CGC....ACCC.GT...C.TG.TCCTT..G....C.T..CAG.......CA.TA.AGG.C. biwaprimus .......T.G...T.GCGCA.C....GT..G..T....C..AG....AC....C..TA.G..T....A.A.G.

Cytb 111111111111112222222222222222222333333333334444444455555555566666666 111122333555667700333344666899012344455556678899900122445578112236780113467992344567810349281472585736370369583691692105147057923439028156286958082847643981786242541162435

C. tentans Yak TCAATTATTCAATGACTCTCTTGCTGAATGTTCCTCTCATATATAATATTTTCCTTTACATTAAACATCTTTCTAGTCTTCCTTATC. tentans Alt ...........T.................A........................................................C. tentans Semi ?............C.........A??????????????????????????????????????????????????????????????C. tentans Stoc ..............................................................................C.......C. tentans Lund ..................C......A...A........................................................C. dilutus FND CT...GTACT.T......CTCCAT.A..CA..TT...T...CG......C..TT...CT.CCGG......CC...TCT.C.TCA..C. dilutus HND CT...GTACT.T......CTCCAT.A..CA..TT...T...CG......C..TT...CT.CCGG......CC....CT.C.TCA..C. dilutus CMN ?TT...GACT.T...T..CTCCATAA.GCAC.TT..CT...CG......C...T.A.CT.CCGG......CC...TCT.C.TCA..C. dilutus WCA CT...GTACT.T......CTCCAT.A..CA..TT...T...CG......C..TT...CT.CCGG......CC...TCT.C.TCA..C. dilutus LMA CT...G.ACTGC......CTCCAT.A..CA..T....T...CG......C..TTC.CCT.CCG.G..C..CC....CT.CTT.A..C. pallidivittatus Yak ?..G.....T.....T.........A.G.A.....T.............G....G...C...C........C.....T........C. pallidivittatus Alt ...GA....T.....T.........A.G.A.....T..........GG.G....G...C..GC........C.....T.C......C. pallidivittatus Omsk ...G.....T.....T..C......A.G.A.....T......................CC..C........C.....T........C. pallidivittatus Lund ...G..G..T.....T..C........G.A.A...T.............G........C...C.......TC....CT........C. pallidivittatus FND C....CGACTGT.....T.TCAAT.A..CA.ATT...T...CGC..A..CCC.T...CT.CCGG......CC...TCT.C.TCA..C. pallidivittatus CND CTT..CGACTGT.....TCTCAATAA..CA..TT...T...CG......C..TT...CT.CCGG......CC...TCT.C.TCA..C. biwaprimus CT.......T.TA.GTC..TCAAT.AT.CA..TTC..TGCG.......C......C......G..TG.....TCG.CT...T..GC


Mitochondrial gene flow in Camptochironomus 391

© 2002 The Royal Entomological Society, Insect Molecular Biology, 11, 387–397

haplotypes. This relationship is reversed in the combinedmt network (Fig. 3B). However, it must be remembered thatall these connections fall outside the 95% limit of reliability.The designated primitive haplotypes unfortunately do notclarify the direction of migration of these populations.C. tentans Lund in the gb2b network supports the view ofearlier studies (Acton, 1962; Kiknadze et al., 1996, 1998)that Nearctic populations of these species arose bymigrated from the Palearctic, but this is contradicted by themt network, where C. dilutus FND is considered primitive.The occurrence of mt gene flow, as outlined below, mayobscure the true origins of the mt haplotypes.

Mitochondrial gene flow

As outlined above, there was an unexpected difference inthe phylogenetic relationships implied by the nuclear andmt sequence data. The tree based on gb2b sequences

Figure 1. Consensus NJ tree inferred from gb2b nt sequence data. Bootstrap values (NJ/MP/ML) are based on 1000 replications. Scale represents Tamura-Nei distance.

Tabl

e 3.

Cor

rect

ed p

airw

ise

ntie

lcot

ide

dist

ance

s ca

lcul

ated

with

Tan

itira

-Nei

met

hod:

gb2

b da

ta (

abov

e di

agon

al)

and

C01

-1 C

ytb

data

(be

low

dia

gona

l)

12

34

56

78

910

1112

1314

1516

1718

1920

2122

23

1. C

. ten

tans

Ger

m0.

003

0.00

70.

003

0.00

30.

014

0.02

80.

014

0.01

70.

014

0.01

70.

014

0.05

30.

052

0.05

3N

/A0.

057

0.04

90.

053

0.04

60.

053

0.13

30.

197

2. C

. ten

tans

Lun

dN

/A0.

003

0.00

00.

000

0.01

00.

025

0.01

00.

014

0.01

00.

014

0.01

00.

049

0.04

90.

050

N/A

0.05

30.

046

0.04

90.

042

0.04

90.

128

0.20

13.

C. t

enta

ns S

toc

N/A

0.00

30.

003

0.00

30.

014

0.02

80.

014

0.01

70.

014

0.01

70.

014

0.05

30.

052

0.05

3N

/A0.

057

0.04

90.

053

0.04

60.

053

0.13

30.

206

4. C

. ten

tans

Sem

iN

/A0.

009

0.00

70.

000

0.01

00.

025

0.01

00.

014

0.01

00.

014

0.01

00.

049

0.04

90.

050

N/A

0.05

30.

046

0.04

90.

042

0.04

90.

128

0.20

15.

C. t

enta

ns A

ltN

/A0.

006

0.00

60.

007

0.01

00.

025

0.01

00.

014

0.01

00.

014

0.01

00.

049

0.04

90.

050

N/A

0.05

30.

046

0.04

90.

042

0.04

90.

128

0.20

16.

C. t

enta

ns Y

akN

/A0.

005

0.00

40.

009

0.00

50.

021

0.00

70.

010

0.00

70.

010

0.00

70.

053

0.05

30.

057

N/A

0.06

10.

053

0.06

00.

053

0.06

00.

133

0.20

67.

C. t

enta

ns A

AK

N/A

N/A

N/A

N/A

N/A

N/A

0.02

10.

025

0.02

10.

025

0.02

10.

047

0.04

70.

050

N/A

0.05

40.

043

0.05

00.

046

0.05

40.

120

0.17

98.

C. d

ilutu

s LM

AN

/A0.

060

0.06

30.

073

0.06

10.

061

N/A

0.00

30.

000

0.00

30.

000

0.05

30.

053

0.05

7N

/A0.

061

0.05

30.

060

0.05

30.

060

0.13

30.

206

9. C

. dilu

tus

CM

NN

/A0.

069

0.07

30.

089

0.07

00.

071

N/A

0.03

60.

003

0.00

70.

003

0.05

60.

056

0.06

1N

/A0.

064

0.05

70.

064

0.05

70.

064

0.13

70.

201

10. C

. dilu

tus

WC

AN

/A0.

059

0.06

30.

073

0.06

10.

061

N/A

0.02

40.

018

0.00

30.

000

0.05

30.

053

0.05

7N

/A0.

061

0.05

30.

060

0.05

30.

060

0.13

30.

206

11. C

. dilu

tus

FN

DN

/A0.

062

0.06

50.

075

0.06

20.

063

N/A

0.02

20.

016

0.00

30.

003

0.05

60.

056

0.06

1N

/A0.

064

0.05

70.

064

0.05

70.

064

0.13

70.

201

12. C

. dilu

tus

HN

DN

/A0.

060

0.06

30.

075

0.06

00.

062

N/A

0.02

10.

016

0.00

40.

001

0.05

30.

053

0.05

7N

/A0.

061

0.05

30.

060

0.05

30.

060

0.13

30.

206

13. C

. pal

lidiv

ittat

us G

erm

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

0.00

70.

017

N/A

0.02

10.

031

0.02

10.

021

0.02

80.

116

0.19

314

. C. p

allid

ivitt

atus

Lun

dN

/A0.

020

0.02

00.

019

0.02

00.

020

N/A

0.06

30.

070

0.06

20.

065

0.06

3N

/A0.

017

N/A

0.02

10.

031

0.02

10.

021

0.02

80.

116

0.19

215

. C. p

allid

ivitt

atus

Om

skN

/A0.

016

0.02

00.

023

0.02

00.

019

N/A

0.05

60.

064

0.05

90.

060

0.05

8N

/A0.

010

N/A

0.00

30.

021

0.00

30.

007

0.02

10.

120

0.19

416

. C. p

allid

ivitt

atus

Alt

N/A

0.02

30.

024

0.02

40.

024

0.02

2N

/A0.

065

0.07

20.

064

0.06

60.

065

N/A

0.01

20.

012

N

/AN

/AN

/AN

/AN

/AN

/AN

/A17

. C. p

allid

ivitt

atus

Yak

N/A

0.01

60.

018

0.01

90.

018

0.01

6N

/A0.

061

0.07

00.

062

0.06

40.

062

N/A

0.00

70.

008

0.00

80.

024

0.00

70.

010

0.02

50.

124

0.19

918

. C. p

allid

ivitt

atus

JN

DN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/AN

/A0.

024

0.02

80.

028

0.12

40.

194

19. C

. pal

lidiv

ittat

us C

ND

N/A

0.06

70.

070

0.08

50.

067

0.06

8N

/A0.

027

0.02

10.

012

0.01

00.

010

N/A

0.06

90.

064

0.07

10.

067

N/A

0.00

30.

021

0.12

00.

192

20. C

. pal

lidiv

ittat

us F

ND

N/A

0.06

80.

069

0.07

70.

067

0.06

7N

/A0.

029

0.02

60.

015

0.01

20.

012

N/A

0.06

70.

064

0.07

00.

068

N/A

0.01

20.

021

0.11

60.

190

21. C

. biw

aprim

usN

/A0.

051

0.05

20.

059

0.05

10.

051

N/A

0.06

60.

071

0.06

10.

065

0.06

3N

/A0.

054

0.05

30.

057

0.05

1N

/A0.

067

0.06

70.

124

0.19

722

. C. o

ppos

itus

N/A

0.15

20.

154

0.16

80.

154

0.15

5N

/A0.

160

0.15

80.

159

0.15

90.

157

N/A

0.15

40.

152

0.15

80.

154

N/A

0.15

70.

149

0.16

40.

155

23. C

. thu

mm

iN

/A0.

165

0.16

70.

166

0.16

40.

166

N/A

0.16

20.

161

0.16

30.

160

0.16

1N

/A0.

168

0.16

80.

170

0.16

9N

/A0.

160

0.15

90.

171

0.17

4


392 J. Martin, V. Guryev and A. Blinov


supports the commonly accepted view on the relationshipsof the Camptochironomus species based on morphologicaland cytological data (Hein & Schmulbach, 1971; Kiknadzeet al., 1998). To better assess the cytological data, a phen-ogram was constructed for populations of these species forwhich cytological data was available (note that other cyto-logically studied NA populations of C. pallidivittatus haveidentical characters to those of CND). Figure 4 shows theconsensus tree from the three trees produced. With theexception that C. biwaprimus is basal to both C. tentans(s.l.) and C. pallidivittatus, and that C. tentans (AAK) islinked to C. dilutus with low bootstrap support, the samplerelationships are consistent with those of the phylogenetictree obtained for gb2b sequence data (Fig. 1). In particular,all C. pallidivittatus samples and all C. tentans (s.l.)samples are grouped together. On the other hand, the ntsequences of the mt genes indicate unexpected inter-specific similarity within both Nearctic and Palearcticpopulations of C. tentans (s.l.) and C. pallidivittatus (Fig. 2).The problem is greatest in the Nearctic branch, whereC. pallidivittatus shows the same level of difference (1.1–2.9%) from the East and West Canadian races of C. dilutusas do these races from each other. Moreover, even removalof all North American C. pallidivittatus from the analysisdoes not produce trees of corresponding topology from the

gb2b and mt sequences. The tree based on gb2bsequences still places C. tentans (s.l.) and C. pallidivittatuson separate branches, while the tree based on mtsequences shows Nearctic populations of C. dilutus inone cluster and all Palearctic populations, regardless ofwhether they are C. tentans (s.s.) or C. pallidivittatus, in aseparate cluster.

Estimation of separation times

A further question of interest is the estimates of time thedifferent populations have been isolated and whether thisallows any conclusions to be drawn about the timing andnumber of colonizations of the Nearctic. In previous studies(e.g. Kao et al., 1994; Chen et al., 1995; Case et al., 1997;Gruhl et al., 1997; Guryev et al., 2001) a separation time of60 My for the separation of the globin genes of C. thummiand C. tentans has been used as the basis for calculatingseparation times of other species. This figure was derivedfrom the Goodman et al. (1988) estimation of the molecularclock for the invertebrate globin sequences. Chen et al.

Figure 2. Consensus NJ tree inferred from Cytb and COI nt sequence data. Bootstrap values (NJ/MP/ML) are based on 1000 replications. Scale represents Tamura-Nei distance. Figure 3. Haplotype networks of the relationships between species and

populations of Camptochironomus species based on (A) gb2b sequences, and (B) mitochondrial sequences, redrawn from output of TCS1.13. Bars represent intermediary haplotypes, while numbers indicate the number of intermediary haplotypes where the lines could not be drawn to scale. Note that C. tentans Lund in (A), and C. dilutus FND in (B),were identified as the primitive haplotype. The 95% confidence limit was seven steps in (A) and fifteen steps in (B).




(1995) also used the rate of change for slowly evolvinggenes in Drosophila of 8 × 10−9/silent site/year (Sharp & Li,1989) and obtained an essentially similar figure (57 My). Bymeans of these values, the separation time estimates werecalculated using corrected distances obtained for gb2bsequences with a substitution rate of 0.335% per My (Table 4).

Data from other insects for the divergence rate of COIand Cytb sequences, where some independent calibration

was available, indicated divergence to be 1.2–1.6% per My(Fleischer et al., 1998; Caccone & Sbordoni, 2001; Farrell,2001). These data also suggested that COI evolved moreslowly than Cytb, whereas our divergence calculations (seeabove) indicated that the two genes have very similar ratesof evolution. Furthermore, the use of any of these valueswill apparently underestimate separation times in the caseof Camptochironomus populations because there is astrong disagreement with the Goodman et al. (1988) andChen et al. (1995) estimates of the C. thummi /C. tentanssplit. This can be illustrated by comparing the mt gammaTNdistance value for C. thummi /C. tentans (d = 0.188) withthat of the beetle Speonomus lostiai and other species ofSpeonomus from which it separated 29 Mya (d = 0.780)(Caccone & Sbordoni, 2001). We therefore used a valueof 6.3–8.4 Mya for the C. biwaprimus /C. pallidivittatus split,as suggested by the separation times obtained from gb2bsequences, to calibrate the molecular clock for the mt data.Thus, the substitution rate used to calculate separationtimes on mt data was 0.75% (corrected distance) per My,which is in good agreement with the data of Su et al. (1998)for the ND5 gene in Japanese beetles. Even this figure maybe an overestimate when one notes that the TN distancevalues of the mt sequences of Camptochironomus are onlyslightly higher than those of gb2b (Table 3), i.e. these mtdivergence times may still be an underestimate of theseparation times.

Discussion

Species with a Holarctic distribution have been of interestas a model for investigation of intraspecific polymorphismand the first steps of the speciation processes. Populationsin Eurasia and North America have been isolated at leastsince the last ice age, and probably longer, with differentia-tion of NA populations postulated to have occurred inSouthern refugia during the ice ages (Howden, 1966). Thisis assumed to have been the situation in the Camptochi-ronomus species (Acton & Scudder, 1971). Kiknadze et al.(1996, 1998) noted that C. tentans (s.l.) had differentiatedcytologically much more on the two continents than hadC. pallidivittatus. However they were unable to determinewhether this difference was due to different times of colon-ization of the New World or to different selective regimes in

Figure 4. Consensus MIX tree based on cytological characters of the Camptochironomus populations, rooted on Chironomus oppositus and C. thummi. The numbers at the nodes indicate the frequency (as a percentage) with which that particular branch was found in the three equal length trees produced in the MIX analysis.

Table 4. Separation time estimates among Nearctic (NA) and Palearctic (PA) samples using gb2b data (above diagonal) and mt (below diagonal)

C. tentans (s.s.) PA C. dilutus NA C. pallidivittatus PA C. pallidivittatus NA C. biwaprimus

C. tentans (s.s.) PA 2.1–5.0 14.6–18.2 12.5–18.2 14.6–17.9C. dilutus NA 7.9–11.9 15.8–19.1 15.8–19.1 17.9–19.1C. pallidivittatus PA 2.1–3.2 7.5–9.6 0.9–9.3 6.3–8.4C. pallidivittatus NA 8.9–11.3 1.3–3.9 8.5–9.5 6.3–8.4C. biwaprimus 6.8–7.9 8.1–9.5 6.8–7.6 8.9




the two species. Indication that the selection regimes maybe different is given by the observation that the C. tentans-like forms are more common than C. pallidivittatus through-out their range and particularly in the Nearctic whereC. pallidivittatus appears to be restricted to the Midwest inSouth Dakota, North Dakota and Saskatchewan.

One important aspect concerns the extent to whichspeciation has proceeded between the various membersof this group. C. tentans (s.l.) and C. pallidivittatus havelong been accepted as separate sibling species (Palmen &Aho, 1966), and the support for this view by the nuclearmolecular data will be considered further below.C. biwaprimus (the only Camptochironomus species recordedin Japan) was considered more closely related to C. tentans(s.s.) on the basis of adult morphology (Sasa & Kawai,1987). Its cytological relationships are ambiguous becausethe chromosome sequences are ones that are basalto both C. tentans (s.l.) and C. pallidivittatus (Fig. 4).However, from the molecular data it appears to be moreclosely related to C. pallidivittatus, because it forms aninternal branch within the C. pallidivittatus cluster (Fig. 1).This suggests that it has retained more primitive chromo-some sequences, which have since been lost fromC. pallidivittatus. Consequently their genetic isolation ismuch more obvious from morphology and cytology thanfrom molecular data, while the latter indicates the relation-ships between the species more accurately. The moleculardata are also consistent with Shobanov et al.’s (1999)separation of the Nearctic and Palearctic populations ofC. tentans (s.l.) into C. dilutus and C. tentans (s.s.), respect-ively. However, the molecular data do not resolve whetherthe Alaskan sample belongs in C. tentans (s.s.) orC. dilutus, but suggest that it is basal to both (Fig. 1).

Population variability and separation time estimates

The mt sequences indicate greater variability between theNA samples of each species than between the Palearcticsamples. This may indicate that there is no real isolation ofthe Palearctic populations across the continent, perhapsbecause gene flow has been occurring for a longer timethan in Nearctic populations, which are postulated to havecome back into contact more recently (Acton & Scudder,1971). The gb2b sequences are also consistent with thisinterpretation.

Several conclusions can be made from separation timeestimates based on gb2b data (Table 4). First of all, the ageof the C. tentans (s.l.)/C. pallidivittatus split that is proposedto have occurred in the Miocene, is about 13–15 Mya.Although palaeomagnetic evidence also suggests 15 Myafor the split of ancient Japan from Eurasia (Su et al., 1998),the isolation of C. biwaprimus appears to have becomecomplete only about 7 Mya, possibly due to continuingmigration because it is a flying insect. Divergence estim-

ates show that intraspecific transcontinental separation is

likely to have been during the Pliocene. Estimates forC. tentans (s.l.) give a range of 2.1–5.0 (Table 4) which,when the standard error is taken into account, suggeststhat divergence had begun at least 2.5 Mya. Our resultssuggest that C. pallidivittatus has been in NA at least aslong as the progenitors of C. tentans (s.l.). If a similar periodof separation is confirmed, then the greater cytologicaldivergence of C. tentans (s.l.) in North America is probablydue to the action of selection.

The time estimates obtained suggest that the isolation ofthe Nearctic and Palearctic populations of the Camptochi-ronomus species has been longer than assumed by pre-

vious studies, which suggested they have been separatedonly since the end of the last ice age (Acton, 1962;Kiknadze et al., 1996, 1998). It is noteworthy that theestimations of intercontinental divergence are of the sameorder of magnitude as those obtained from mtDNA data forNA songbirds and their Palearctic relatives (Klicka & Zink,1997, 1998). These authors correlate the genetic isolationwith enlargement of the Northern Hemisphere ice sheetsabout 2.4 Mya, in the Pliocene. Thus, it seems likely thatinsects, like the vertebrates, began on the path to specia-tion in the Nearctic before the late Pleistocene glacialcycles.

Interspecific introgression of mt DNA

The gb2b (Fig. 1) and mt (Fig. 2) trees are not congruent.Therefore we need to identify the source of the incongru-ence, if possible, and determine which tree more accuratelyrepresents the relationships between the Nearctic andPalearctic populations. Morphology (Beermann, 1955; Hein &Schmulbach, 1971), and cytology (Fig. 4) are both consist-ent with the tree based on the gb2b sequences. There isfurther support for this tree from another nuclear gene, thessp160 gene (Makarevich et al., 2000). This gene iscomplete in C. pallidivittatus, in both the Nearctic andPalearctic, and in C. biwaprimus, while C. tentans (s.l.) fromeither continent contain a deletion of a large part of thegene (Makarevich et al., 2000; S. T. Case, personal com-munication). It seems unlikely that such a deletion aroseindependently in each region, but rather that it occurredafter the separation of C. tentans (s.l.) from the commonancestor of C. pallidivittatus and C. biwaprimus, and wascarried to the Nearctic region by the C. tentans (s.l.) migrantsthat eventually became C. dilutus.

We can therefore conclude that it is the mt data thatare responsible for the lack of congruence. Severalmechanisms have been proposed to account for a lack ofcongruence in phylogenies based on mt sequence data(Brower et al., 1996). While we cannot completely rule outmt lineage sorting as an explanation, we would favour anexplanation based on mt gene flow.

Takahata & Slatkin (1984) proposed a model for the pro-

cess of gene flow, with two components very important to




its occurrence: species must hybridize in nature and live ina region of sympatry. This is true for the Holarctic speciesin this study, because C. pallidivittatus is commonly foundin the same pools as C. tentans (s.l.), but at low frequencies(Kiknadze et al., 1998). The only evidence of natural hybrid-ization between C. tentans (s.l.) and C. pallidivittatuscomes from Beermann (1955) who found one hybrid in hiscytological studies of German populations. Laboratoryhybridization is possible, however, between populations ofC. tentans (s.l.) and C. pallidivittatus from both continents(Beermann, 1955; Tichy, 1970; Hein & Schmulbach, 1971).For Nearctic populations it was shown that when the femalewas C. pallidivittatus all hybrid egg masses were infertile, butif the female was C. dilutus hybrid egg masses had someviability, although it was extremely low (Hein & Schmulbach,1971). In Europe, C. tentans (s.s.) and C. pallidivittatus canproduce hybrids in both directions (Beermann, 1955).If these results are indicative of the hybridization eventsresponsible for the gene flow, we would expect thedirection of the flow to be from C. dilutus to C. pallidivittatusin the Nearctic, while in the Palearctic it could be in eitherdirection.

The estimates of divergence time based on mt data(Table 4) may provide evidence relevant to the direction ofgene flow, even though they have little meaning in termsof phylogeography. The mt data show that maximal valuesof the transcontinental comparisons are similar tothose obtained for comparisons of C. tentans (s.l.) andC. pallidivittatus. This would be consistent with the mtDNAof different species having been fixed on each continent. Inthe Nearctic it seems likely that mt gene flow has occurredfrom C. tentans (s.l.) into C. pallidivittatus, because thehaplotype relationships (Fig. 3B) indicate that those ofC. pallidivittatus are derived from those of C. dilutus. This iswhat might be expected because C. pallidivittatus has alimited distribution in NA compared to C. dilutus, and there-fore its mtDNA could be wiped out by gene flow. It is alsoconsistent with the direction of gene flow expected fromlaboratory hybridization. If the mtDNA of C. tentans (s.l.) hasbecome fixed in the Nearctic, we suggest that the oppositeoccurred in the Palearctic and the mtDNA of Palearcticspecimens is descended from C. pallidivittatus. The slightlylower transcontinental values for the mt data compared tothose from gb2b data (Table 4) may be due to mt gene flowin the early evolutionary stages of these species. The smallintracontinental divergence indicates that mt gene flowcontinued after the continents become isolated.

The conclusion that the incongruence of the mt data andthe other data is due to gene flow rather than lineage sort-ing is supported by data for another pair of Holarctic siblingspecies, Chironomus entis and C. plumosus (Guryev &Blinov, 2002). These species can also produce interspecifichybrids with each other (Kiknadze et al., 2000). The pheno-

grams show the same incongruence as that seen in the

Camptochironomus group, with ‘geographical’ relation-ships for the mt sequences and ‘specific’ relationshipscongruent with the morphological and cytological data forthe nuclear sequences. It seems unlikely that both groupsshow this similar pattern as a result of lineage sorting,whereas the presence of low levels of hybridization in bothgroups suggests gene flow as a more likely cause. Thisdoes not mean that mt gene flow will be common amongstChironomus species, as our previous studies (Makarevichet al., 2000; Guryev et al., 2001) have revealed no obviousinconsistencies between mt and nuclear sequence data.There is also no evidence of mt gene flow between the twocryptic species included under the name C. calligraphus(Spies et al., 2002). However these species show 22%sequence difference in the mt COII gene, so may havebeen genetically isolated for several million years, althoughfound in the same pools today. It would not be surprising,however, for further examples of mt gene flow resultingfrom hybridization to be found in other recently arisensibling chironomid species.

Experimental procedures

Specimens of C. tentans (s.l.), C. pallidivittatus and C. biwaprimus wereobtained from natural populations or laboratory stocks of knowngeographical origin (Table 1). Two species of the subgenusChironomus, namely C. thummi (syn. C. riparius) and C. opposituswere used as outgroup species.

Total DNA for each population has been isolated from ten larvaeaccording to the protocols described in Guryev et al. (2001). Thepooling of the DNA took place before it was known that this mightlead to problems during PCR amplification. However, there is noindication that our results have been adversely affected. In thecase of C. pallidivittatus CND, we were able to sequence threelarvae separately and found that there were substitutions at only0.14% of sites, and these made no difference to the position of thesample in the trees.

We analysed portions of two mitochondrial genes, Cytochromeb (Cytb) and Cytochrome-c oxidase subunit I (COI ), and thenuclear globin 2β (gb2b) gene. We amplified and sequenced691 bp from Cytb, 614 bp from COI, and 300 bp from gb2b, usingthe methods for PCR amplification and sequencing as describedin Guryev et al. (2001).

Nucleotide sequence accession numbers in GenBank for Cytbsequences are AF109698–AF109713; for COI are AF110155–AF110166; and for gb2b sequences AF110167–AF110175,AJ003799, −802, −808, −810.

Nt sequences were aligned manually with no ambiguities.These alignments were used for construction of trees based onthe NJ, MP and ML methods implemented in the PHYLIP 3.5(Felsenstein, 1993) and MEGA 2.1 (Kumar et al., 2001) packages.The ML analysis applied a Ti / Tv ratio of 3.0, the value determinedfrom an MP analysis using the procedure recommended in PAUP*.Statistical support for the trees was evaluated from bootstrapping(1000 replications) (Felsenstein, 1985). The phylogenetic treesobtained by NJ, MP and ML methods were congruent for each dataset, therefore only NJ consensus trees are presented in each casewith bootstrap values specified for NJ, MP and ML methods.




Consensus between the mt and gb2b trees was tested usingthe Templeton non-parametric test (Templeton, 1983) and theShimodaira-Hasegawa (SH) test (Shimodaira & Hasegawa, 1999),as implemented in PAUP* Version 4.0b10 (Swofford, 1998). TNdistances (Tamura & Nei, 1993) were calculated using theMEGA 2.1 package (Kumar et al., 2001). These distances werelinear in a saturation plot (Guryev et al., 2001) and gave the sametree topologies as those obtained using gammaTN distances, sothey were preferred because of the lower associated standarderrors. Haplotype networks for the sequence of each populationwere generated using TCS1.13 (Clement et al., 2000). A cytolog-ical tree was constructed using MIX, as in Guryev et al. (2001),with fifty characters: arm combination (3), basic sequences of armsA (24), E (8), F (8), and position of nucleolus (7). The data wereobtained from Martin (1979), Wülker et al. (1989), Kiknadze et al.(1996, 1998), and unpublished data of J.M.

Acknowledgements

The study was partly supported by grants from theAustralian Department of Industry, Science and TechnologyBilateral Science and Technology Support Program and theUniversity of Melbourne Collaborative Research Program(J.M., A.B.); and Russian Foundation for Basic Researchgrant 99-04-49265 (A.B., V.G.). We are grateful to DrM.G. Butler (ND State University, Fargo, USA), and ProfI.I. Kiknadze, Dr Eu. Andreeva (Institute of Cytology andGenetics, Novosibirsk, Russia), Dr K. Kawai (University ofHiroshima, Japan), Prof J.-E. Edstrom (University of Lund,Sweden), and Prof B. Daneholt (Karolinska Institute, Stock-holm, Sweden) for providing specimens from a number ofthe studied populations. We thank Dr B.T.O. Lee (Universityof Melbourne, Australia) and two unnamed referees, forhelpful comments on the manuscript.

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Population variability in Chironomus (Camptochironomus) species (Diptera, Nematocera) with a Holarctic distribution: evidence of mitochondrial gene flow