doi:10.1111/j.1095-8649.2009.02331.x, available online at www.interscience.wiley.com
Phylogeography and sympatric differentiationof the Arctic charr Salvelinus alpinus (L.) complex in
Siberia as revealed by mtDNA sequence analysis
S. S. Alekseyev*†, R. Bajno‡, N. V. Gordeeva§, J. D. Reist‡, M. Power||,A. F. Kirillov¶, V. P. Samusenok** and A. N. Matveev**
*Kolzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26,Moscow 119991, Russia, ‡Fisheries and Oceans Canada, Freshwater Institute, 501 University
Crescent, Winnipeg, Manitoba R3T 2N6, Canada, §Vavilov Institute of General Genetics, RussianAcademy of Sciences, ul. Gubkina 3, Moscow, 119991, Russia, ||Department of Biology, 200
University Avenue West, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada, ¶Instituteof Applied Ecology of the North, Academy of Sciences of the Sakha Republic (Yakutia), pr. Lenina35, Yakutsk 677007, Russia and **Faculty of Biology and Soil Biology, Irkutsk State University,
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INTRODUCTION
Widely distributed polymorphic species are among the most interesting objects ofphylogeographic and microevolutionary studies. The Arctic charr Salvelinus alpinus(L.) complex is one of the most polymorphic and evolutionarily labile fish speciesgroups (Jonsson & Jonsson, 2001). High phenotypic variation throughout the cir-cumpolar range of this species complex and the wide occurrence of sympatric formsdiffering in biology and morphology (Johnson, 1980; Behnke, 1984; Savvaitova,1989, 1995; Jonsson & Jonsson, 2001; Klemetsen et al., 2003 and references therein)are sources of ongoing debate over mechanisms responsible for this diversity; inparticular, the relative importance of allopatric and sympatric modes of evolution.
The study of mitochondrial (mt) DNA control region sequence variation in S.alpinus species complex throughout its wide range provided a global view oncharr phylogeography, revealing five major phylogenetic groups (Brunner et al.,2001). These data, however, were insufficient to clarify the interrelationships ofS. alpinus populations on smaller geographic scales. In particular, the whole ofSiberia was represented by only 14 specimens from six populations in two areas.Subsequent mtDNA studies of S. alpinus and related species (Radchenko, 2003,2004; Oleinik et al., 2004, 2007; Shed’ko et al., 2007) were based mainly on fishfrom the Pacific Basin and included no, or few, S. alpinus from Siberia. Othergenetic studies have examined allozymes (Osinov et al., 1996, 2003; Osinov, 2002),microsatellites (Samusenok et al., 2006) and karyotypes (Alekseyev et al., 1997), butnone have developed a detailed picture of the widely distributed Siberian populationsbecause of their relative remoteness.
Representatives of S. alpinus complex are found continuously along the Arcticcoasts of Siberia; in interior Siberian regions they are distributed in headwater lakesin the Enisei, Khatanga and Pyasina basins in Taimyr; in the Chaya, Vitim, Olekma(the Lena tributaries) and Lake Baikal basins in northern Transbaikalia and in theupper reaches of the Aldan, Yana, Indigirka and Kolyma rivers (Berg, 1948; Kirillov,1972; Savvaitova, 1989; Pavlov et al., 1999; Alekseyev & Kirillov, 2001; Gudkovet al., 2003; Osinov et al., 2003). Among interior regions, Transbaikalia stands outas the southernmost part of the range of the S. alpinus complex in Siberia, as anarea hosting the largest number of surveyed S. alpinus populations, most of themnewly discovered, and as an important centre of S. alpinus polymorphism (Alekseyevet al., 1999, 2000, 2002). Dispersal routes of S. alpinus into and within this area,in particular to the Baikal Basin, are not fully understood (Savvaitova et al., 1977;Alekseyev & Kirillov, 2001). Many Siberian S. alpinus populations are representedby two to three sympatric forms (ecotypes) exhibiting various degrees of divergence,including morphological distinctiveness and reproductive isolation (Savvaitova,1989; Pavlov et al., 1999; Alekseyev et al., 2000, 2002; Romanov, 2003). The wideoccurrence of sympatric forms, coupled with relative isolation, makes the Siberianpopulations of S. alpinus particularly suitable for studying incipient speciation.
Earlier emphasis on allopatric speciation as the only possible mode of speci-ation (Mayr, 1963) has given way to a growing recognition of the importanceof sympatric speciation in evolution (Ritchie & Phillips, 1998). Sympatric speci-ation is supported by theoretical (Kondrashov & Mina, 1986; Turner & Burrows,1995; Dieckmann & Doebeli, 1999; Kondrashov & Kondrashov, 1999) and empir-ical (Schliewen et al., 1994; Turner & Burrows, 1995; Mina et al., 1996; Volpe &
Ferguson, 1996; Gislason et al., 1999; Danley et al., 2000; Barluenga et al., 2006)evidence. Nevertheless, well-documented cases remain scarce and the role of sym-patric speciation in evolution is incompletely understood.
In the earlier studies of Salvelinus spp., both allopatric (Nilsson & Filipsson,1971; Nyman, 1972; Viktorovskii, 1978; Nyman et al., 1981; Klemetsen & Grotnes,1980; Klemetsen, 1984; Romanov, 2003) and, more frequently, sympatric (Ferguson,1981; Klemetsen et al., 1985; Hindar et al., 1986; Riget et al., 1986; Magnusson& Ferguson, 1987; Savvaitova, 1989, 1995; Danzmann et al., 1991; Sandlundet al., 1992; Osinov et al., 1996; Gislason et al., 1999; Jonsson & Jonsson, 2001;Osinov, 2002; Klemetsen et al., 2003) origins of sympatric forms have beensuggested. Genetic data have supported the sympatric divergence of forms in severallakes (Hindar et al., 1986; Magnusson & Ferguson, 1987; Danzmann et al., 1991;Volpe & Ferguson, 1996; Gislason et al., 1999; Osinov, 2002; Wilson et al., 2004).The most comprehensive study provided evidence for both sympatric and allopatricorigins of presently coexisting forms of S. alpinus (Wilson et al., 2004), as shownfor sympatric and allopatric forms of coregonids (Pigeon et al., 1997), anotherpolymorphic group of northern salmonoids.
In this work, the results of an mtDNA-based study of S. alpinus complex in thepoorly studied Siberian part of its range are presented. The goals were to establishthe phylogenetic relationships of its representatives from various Siberian regions,with an emphasis on Transbaikalia, to infer dispersal routes of S. alpinus among andwithin these regions and to investigate possible modes of speciation that may haveled to the origins of sympatric forms.
MATERIALS AND METHODS
S A M P L I N GSalvelinus spp. were sampled with gillnets in 1995–2006 in 49 Siberian and seven
North American water bodies [Table I and Fig. 1(a), (b)]. Sampling locations (latitude,longitude), lake dimensions and elevation are given in Table I. Samples were collectedfrom natural populations without histories of transplantation. Additional information aboutthe Transbaikalian lakes of Siberia appears in Alekseyev et al. (1999, 2002) and Samusenoket al. (2006). Fish from Canoe River, Fraser and Karluk lakes were identified as Dolly VardenSalvelinus malma (Walbaum); those from all other populations as S. alpinus complex. Insamples from Siberian locations, S. alpinus were classified as dwarf, small or large formsusing length-at-age distributions and morphological characters (Alekseyev et al., 2000, 2002;Alekseyev & Kirillov, 2001; Samusenok et al., 2006) or, if the data were insufficient, as‘form unknown’. Fish analysed for mtDNA were sub-sampled from the total samples sothat all sympatric forms were represented. Muscle, fin clip or blood samples were storedin ethanol, in a 20% dimethyl sulphoxide (DMSO) salt solution (muscle, fin clips) or anethylenediaminetetra-acetic acid (EDTA) solution (blood) until DNA extraction.
MTD NA A NA LY S I STotal genomic DNA was extracted from tissue using commercial kits (DNeasy Tissue Kit;
www.quiagen.com) following manufacturer’s instructions. A 655 base pair (bp) fragment,including the entire left domain of the char mtDNA control region, was amplified usingthe forward primer tPro2 (5′-ACC CTT AAC TCC CAA AGC-3′; Brunner et al., 2001) andreverse primer ARCH1 [5′-CC(CT) TGT TAG ATT T(CT)T TCG CTT GC-3′] located in theright domain.
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The PCR profile consisted of an initial denaturation at 95◦ C (4 min) followed by 32 cyclesof 94◦ C (1 min), 50◦ C (1 min), 72◦ C (1·5 min), followed by a final extension at 72◦ C(10 min). Reactions contained 10–60 ng DNA, 0·8 μM of each primer, 1·0 mM MgCl2, 200μM of each dNTP, 1U Taq polymerase (New England Biolabs Inc.; www.neb.com) and 1×PCR buffer. PCR products were purified using a commercial kit (QiaQuick PCR PurificationKit; Qiagen Inc.) following manufacturer’s instructions. A set of 546 samples was sequencedfor 507 bases with nested backwards primer Char3 (5′-CCC TAT GCA TAT AAG AGA ACGC-3′). Direct sequencing of the control region PCR product was performed using dRhodamine,or BigDye 3.1 Terminator Cycle Sequencing Ready Reaction kits (Applied Biosystems;www.appliedbiosystems.com) following manufacturer’s instructions using a primer annealingtemperature of 52◦ C. The resulting sequence reaction products were subsequently purifiedwith a commercial kit (DyeEx 2.0 Spin Kit; Qiagen Inc.) and analysed on an ABI3100 GeneticAnalyzer (Applied Biosystems).
Sequences were aligned using Seqscape 2.5 (Applied Biosystems), and a haplotype foreach sample was assigned and identified following the naming convention of Brunner et al.(2001), in which an area abbreviation (e.g. SIB = Siberia) and a number (e.g. 1) are combinedto designate a unique haplotype. Haplotypes designated in previous publications are indicatedin normal typeface (e.g. SIB1), haplotypes observed in this study in bold typeface (e.g. SIB15)and haplotypes observed in this study that corresponded to those of Brunner et al. (2001) areboth bold and underlined (e.g. SIB5). Each haplotype sequence revealed in this study wasverified by sequencing one representative sample in the reverse direction with primer tPro2.
Selected haplotypes reported by Brunner et al. (2001) (550 bp) were retrieved fromGenbank and added to the analysis. The analysis was carried out using the 501 bp fragmentoverlapping the sequences obtained in this study and the external sequences, for whichinitial haplotype designations were retained. Sequences were analysed with MODELTEST3.7 (Posada & Crandall, 1998) to determine the substitution model that best fits the sequences.The most appropriate model, as chosen by the Akaike information criterion (Akaike, 1974),was the HKY85 (Hasegawa–Kishino–Yano 85; Hasegawa et al., 1985) model, plus I + �.The phylogenetic relationships among the haplotypes were estimated with a maximumparsimony (MP) and a neighbour-joining (NJ) tree, based on maximum likelihood distances(HKY85 + I + � model) using PAUP* 4b10 (Swofford, 1998). Additionally, NJ treesbased on other models [HKY, Tamura & Nei (1993), Kimura’s 2-parameter, maximumcomposite likelihood] were constructed using PAUP* 4b10 and Mega 4.0 (Tamura et al.,2007). Bootstrap analysis (Felsenstein, 1985) was used to estimate support for the resultingtopologies with 1000 replicates. The haplotype network was constructed with the medianjoining algorithm (Bandelt et al., 1999) using Network 4.1.1.2. Genetic diversity withingroups was measured as haplotypic and nucleotide diversity (Nei & Tajima, 1981; Nei, 1987);an AMOVA (analysis of molecular variance) was performed to estimate the proportion ofgenetic diversity within and among population groupings in Siberia (Arlequin 3.0, Excoffieret al., 2005). The AMOVA was designed to calculate the amount of genetic variance, firstamong four Siberian regions, among basins or their parts within these regions, and thenwithin basins. The regions were defined as Transbaikalia (Baikal, Chaya, Vitim, Olekmabasins), Taimyr (Pyasina, Khatanga, Khatanga Bay basins), mountains of eastern Siberiabeyond Transbaikalia (Aldan, upper–middle Yana, Indigirka, Kolyma basins) and the coastalzone of eastern Siberia (Olenek Bay, Lena lower reaches and delta, Yana Delta, Indigirka Deltabasins). AMOVA was also used to estimate genetic diversity among lakes with polymorphicpopulations of S. alpinus and among and within sympatric forms within these lakes. Inaddition to populations 1–49 (Table I), three Taimyr populations, studied by Brunner et al.(2001), from lakes Lama (Pyasina Basin, two sympatric forms), Ayan (Khatanga Basin) andArilakh (Khatanga Basin) (eight individuals) were used for AMOVA and to estimate geneticdiversities. Genetic homogeneity between sympatric forms in populations with more than asingle haplotype was tested using Fisher’s exact test or a Monte-Carlo randomization processwith 1000 iterations using the program CHIRXC (Zaykin & Pudovkin, 1993). New sequenceswere deposited in GenBank under accession numbers EU310898–EU310926.
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RESULTS
S E Q U E N C E VA R I AT I O N
Sequencing revealed 33 variable positions defining 29 haplotypes (Table Iand Fig. 2), which differed by 1–17 substitutions (0·20–3·4% divergence). Twohaplotypes, SIB5 {Lakes Krugloe, Rys’, Kungesalakh, Novaya R. in Taimyr [30–33,here and below numbers in square brackets correspond to locality numbers in Table Iand Fig. 1(a), (b)]} and SIB8 (Lakes Bol’shoi Namarakit [10] in Transbaikalia andUlakhan-Silyan-Kyuel’ [40] in the Yana Basin) were identical, within the overlapping501 bp fragment, to SIB5 and SIB8 in Brunner et al. (2001), respectively. HaplotypeBER12, within the overlapping 485 bp fragment, corresponded to BER21 reportedin Shed’ko et al. (2007). Haplotypes reported by Brunner et al. (2001) from threeTransbaikalian lakes (Bol’shoe Leprindo [23], Gol’tsovoe [21] and Frolikha [1])(SIB6, SIB4 and SIB8, respectively) and from Floods Pond (Maine) [56] (ACD4)did not match the new haplotypes in this study in the same localities (SIB21 in thefirst two lakes, SIB10 in the third, ACD9 in the fourth), differing from them by oneto two substitutions.
P H Y L O G E N E T I C R E L AT I O N S H I P S O F H A P L OT Y P E S
The NJ (Fig. 3) and MP (not shown) bootstrap consensus trees had similartopologies. Both included clusters of haplotypes with 59–93% bootstrap support thatcorresponded to the Arctic, Bering, Atlantic and Acadia groups, sensu Brunner et al.(2001). The two latter groups, along with haplotypes from Siberia, form a cluster with42–46% support that corresponded to the Atlantic + Siberia + Acadia supergroup(ASA) of Brunner et al. (2001). Haplotypes from Siberia, however, do not forma single cluster corresponding to the Siberia group sensu Brunner et al. (2001).SIB1, SIB2, SIB3, SIB5 and SIB9 clustered together with the Atlantic group, as didSIB26 in the NJ tree (in the MP tree, it was basal to the Acadia + Atlantic + SIB1-3, 5, 9 cluster). The remaining Siberian haplotypes, with the exception of SIB29,formed a separate group. Haplotype SIB29 was basal to the whole ASA cluster.Neither of the two trees provided high bootstrap support for clusters of Siberianhaplotypes or their combinations with other ASA haplotypes. Topologies of treesbased on other substitution models differed slightly from the NJ tree shown inFig. 3. Haplotypes SIB1, SIB2, SIB3, SIB5, SIB9 connected to the Atlantic + Acadia
FIG. 1. (a) Sampling sites of this study (circles) in Siberia and North America (inserts). Sites sampledby Brunner et al. (2001) in Taimyr and Transbaikalia are added as squares. Association of sites withthe phylogenetic groups of Salvelinus spp. haplotypes reported by Brunner et al. (2001): black circles,Bering group; black circles with white center, Arctic group; other figures, Atlantic + Siberia + Acadiasupergroup (white and grey circles and squares, Siberia group, dotted circle, Acadia group). Associationof sites with subgroups of the Eurasian group recognized in this study: white circles and square, EastSiberian subgroup; grey, Eurosiberian subgroup. Refer to text for details. (b) Detailed map of the locationof studied populations in Transbaikalia, with indication of the control region mtDNA haplotypes foundin this study. The range of Salvelinus alpinus in Transbaikalia is shown in grey, with broken linesindicating boundaries between lake and river basins. I, Baikal Basin, II, IV, Lena Basin; II, Chaya Basin;III, Vitim Basin; IV, Olekma Basin (a, Chara Basin, b, Khani Basin). Whole circles, circles with two andthree sectors correspond to populations in which respectively one, two and three haplotypes were found.Population numbers in (a) and (b) correspond to those in Table I.
groups and, in some trees, were monophyletic; SIB26 and SIB29 tended to fallinto the main Siberian cluster. In the haplotype network of ASA (Fig. 4), Siberiaand Atlantic haplotypes were closely grouped, SIB1, SIB2, SIB3, SIB5 and SIB9being intermediate between other Siberian and Atlantic haplotypes. The Acadiahaplotypes were well separated from both Atlantic and Siberia haplotypes by atleast six mutational steps (1·2% divergence).
TA X O N O M I C A S S O C I AT I O N S O F H A P L OT Y P E S
Bering group haplotypes BER12 and BER13 were found in fish morphologicallyidentifiable as Dolly Varden (S. malma), whereas all other haplotypes were found infish identifiable as Arctic charr (S. alpinus). Unexpectedly, haplotypes BER10 andBER11 of the Bering group appeared in some Siberian populations of S. alpinus.
G E O G R A P H I C A L D I S T R I B U T I O N O F H A P L OT Y P E S
The only Arctic group haplotype observed in this study, ARC19, and two Beringgroup haplotypes BER12 and BER13 were observed in S. alpinus and S. malmafrom Arctic Canada, respectively. Two other Bering group haplotypes were foundin populations of S. alpinus along the Siberian Arctic coast (BER10) and in theupper Kolyma Basin (BER11). The Acadia group haplotype ACD9 was found inMaine. Other haplotypes of ASA, sensu Brunner et al. (2001), were distributed inSiberia. The upper–middle Indigirka Basin is typified by haplotypes SIB27, SIB29and SIB30; the upper–middle Yana Basin by SIB8, SIB10 and SIB28, the AldanBasin by SIB26 and SIB27, the Vitim Basin by SIB10, SIB12, SIB13, SIB14, SIB19,SIB15, SIB16, SIB17, SIB18 and SIB8, the Olekma Basin by SIB20, SIB21, SIB22,SIB23 and SIB24, the Chaya Basin by SIB10, and the Baikal Basin by SIB10 andSIB11. Haplotype SIB5 was distributed in the Taimyr region (the Pyasina Basin andcoastal populations in the Khatanga Bay) and SIB25 had wide distribution along theArctic coast (Khatanga and Olenek Bays, Lena Delta); these two haplotypes, dif-fering in seven substitutions (1·4% divergence), occurred in the same samples fromthe Khatanga Basin. In the Olenek Basin and Lena Delta, the geographical rangeof SIB25 overlaps with that of BER10 (10 substitution difference, 2% divergence);these two haplotypes are found in sympatry in the first region [Fig. 1(a), (b) andTable I].
In Transbaikalia, the most extensively sampled region, the most widespreadhaplotype SIB10 was found in the Baikal Basin and the adjacent parts of the Lena(Chaya and Vitim) Basin. Haplotype SIB14 was widely distributed in the VitimBasin; SIB21 in the Chara Sub basin of the Olekma Basin and SIB20 in its KhaniSub basin and in one adjacent lake in the Chara Sub basin. Other haplotypes wererestricted to single lakes. Two Transbaikalian lakes contained three haplotypes, threeother lakes contained two and 24 lakes only one haplotype. Sympatric haplotypesdiffered by one to three substitutions (0·2–0·6% divergence), with the exception ofhaplotype SIB17 from Lake Leprindokan, which differed from sympatric SIB15 andSIB16 by five and six mutations (1 and 1·2% divergence), respectively.
Haplotypes observed in Transbaikalia were specific to the region, with theexception of SIB8 and SIB10, which were also detected in the Yana Basin. Likewise,the Aldan and the Indigirka Basins had haplotype SIB27 in common. No haplotypeswere observed in common between Transbaikalia or the upper–middle Yana Basin,
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on the one hand, and the upper–middle Indigirka or Aldan Basins, on the other hand,and within Transbaikalia, between the Vitim and Olekma Basins. Because only SIB5,SIB25 and BER10 were found in the north, no haplotypes were shared betweenpopulations of the upper–middle reaches and the delta regions of the Lena, Yanaand Indigirka Rivers. Haplotypes in the network did not strictly cluster by basin,despite obvious differences in the sets of haplotypes observed in different basins(Fig. 4). Among Siberian ASA haplotypes, SIB10 was most abundant (present in 12of 49 Siberian populations); the network indicated that this haplotype had the highestancestral probability, as it had the largest number of branches (8) radiating from it
to haplotypes in samples from the Transbaikalia, Yana and Indigirka basins, as wellas to haplotypes in the Acadia group (Fig. 4).
D I S T R I B U T I O N O F G E N E T I C D I V E R S I T Y
Among the four Siberian regions, the largest number of haplotypes (16) andthe largest haplotype diversity appeared in Transbaikalia (h = 0·848), and among15 basins, in the Vitim Basin (h = 0·810). The largest nucleotide diversities (π =0·0053–0·0083) were observed in water bodies (Kungasalakh, Novaya, Baganytta-Kyuel’ [32–34], Lama) and basins (Lena Delta, Khatanga and Olenek Bays, Pyasina)that contained haplotypes of different phylogenetic groups (SIB25–BER10) orsubgroups (SIB7, SIB25–SIB5, SIB9) (see below), and also in the basins of theupper–middle Indigirka and of the Vitim (Table II). Notably, estimates of geneticdiversity may be biased in small samples that are not sufficiently representative todetect rare haplotypes or accurately measure haplotype frequencies. The results ofAMOVA indicated that differences between regions, between basins within regionsand within basins explained about equal amounts of the total genetic variance (36,31 and 33%, respectively, in all cases P < 0·001) (Table III).
S Y M PAT R I C F O R M S
Sympatric forms of S. alpinus in 23 lakes (22 in Transbaikalia) were analysed.In each lake, the forms shared one or two haplotypes. No significant localdifferentiation in haplotype composition appeared between sympatric forms in lakeswith multiple haplotypes (Table I), although heterogeneity was close to significance(P = 0·08) in Lake Leprindokan [9]. Sympatric forms in seven lakes (Svetlinskoe[2], Kalarskii Davatchan [7], Leprindokan [9], Bol’shoi Namarakit [10], Irbo
FIG. 2. Mitochondrial DNA sequence variation of the 507 bp fragment from the control region of Salvelinusalpinus analysed in this work (abbreviations correspond to those in Table I). Additional sequencesfrom Brunner et al. (2001) were retrieved from Genbank, changed to the reverse and complementsequence and included in the analysis: ARC3, S. a. taranetzi, Seutakan River, (Chukotka Peninsula);ARC6, S. a. erythrinus, Hall Lake (Melville Peninsula, Canada); ARC7, S. a erythrinus, Nauyuk Lake(Nunavut, Canada); ARC15, S. a. alpinus, River near Isortoq Fjord (Greenland); BER2, S. malma,S. albus, Kamchatka River (Kamchatka Peninsula); BER5, S. malma, Kakhmauri River (ParamushirIsland); BER8, S. malma, Prince William Sound (Alaska); BER9, S. malma, Auke Creek (Alaska);SIB1, SIB2, S. a. alpinus, Kuolimo Lake (Finland); SIB3, S. a. alpinus, Spitsbergen (Norway); SIB4,S. a. alpinus (Putoranchik), Ayan Lake (Taimyr) and S. a. erythrinus, Goltsovoe Lake (Baikal region);SIB6, S. a. erythrinus, Leprindo Lake (Baikal region); SIB7, S. a. alpinus (Pucheglazka), Lama Lake(Taimyr Peninsula); SIB9, S. a. drjagini, Lama Lake (Taimyr Peninsula); ATL1, S. a. salvelinus, S. a.alpinus, several localities in Europe and Greenland; ATL7, S. a. alpinus, Femund Lake (Norway); ATL8,S. a. alpinus, S. a. erythrinus, several localities in Europe and North America; ATL14, S. a. alpinus,Fjellfrosvatnet (Norway); ATL15, S. a. erythrinus, Fraser River (Labrador); ACD2, S. a. oquassa, LacChaudiere (Quebec); ACD4, S. a. oquassa, Floods Pond, Penobscot Lake (Maine), Lac Rond (Quebec);ACD5, S. a. oquassa, Walton Lake (New Brunswick); SVET, Salvethymus svetovidovi, Elgygytgyn Lake(Chukotka Peninsula). Names of Salvelinus taxa and localities for external haplotypes correspond tothose in Brunner et al. (2001). Haplotypes observed in this study are indicated as bold typeface, thoseobserved by Brunner et al. (2001) are indicated as regular typeface and those observed in both studiesare bolded and underlined. Genbank accession numbers are given next to the sequence. The referencesequence (ARC19) is that of S. alpinus from Alexandra Lake (Ellesmere Island, Canada). Base positionsfor variable sites are given. The ‘.’ indicates identity and the ‘-’ indicates presence of a deletion to thereference haplotype at that base position.
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83 8094 93
67 5878 50
Arctic
ARC3ARC6
ARC15ARC7
ARC19
BER10BER12
BER13BER8
BER2
BER9BER5
BER11
72 59Bering
SIB1SIB2
ATL1
ATL8
ATL7
ATL1568 55
74 71
ATL14
SIB3SIB9
SIB26ACD9
ACD4
ACD2
88 93 ACD7ACD5
SIB8SIB18
SIB12SIB13
SIB14SIB17
SIB15SIB16
SIB22SIB23SIB30
SIB25SIB28
SIB7SIB19
SIB4SIB21
SIB24SIB6
SIB27SIB10SIB11
SIB20SIB29
Salvethymus svetovidovi
0.001 substitutions/site
Siberia
Siberia
Siberia
Atlantic
Acadia
SIB5
FIG. 3. Neighbour-joining bootstrap consensus tree of the phylogenetic relationships of Salvelinus alpinuscomplex haplotypes observed in this study including selected Salvelinus spp. haplotypes reportedby Brunner et al. (2001) (abbreviations correspond to those in Table I and Fig. 2). Salvethymussvetovidovi is used as an outgroup. Phylogenetic groups designated by Brunner et al. (2001) are shown.Haplotypes observed in this study are indicated as bold typeface, those observed by Brunner et al. (2001)are indicated as regular typeface and those observed in both studies are bold and underlined. Numbers arebootstrap values (>50%) computed by 1000 replications using maximum likelihood distance measurescalculated using the HKY85 + I + � model of nucleotide substitution followed by maximum parsimonyclustering algorithms.
[14], Severonichatskoe [27] in Transbaikalia; Tunernde [35] in the Aldan Basin)had common unique haplotypes that were restricted to the lake. No haplotypesdistinguished allopatric dwarf, small or large forms in different lakes, nor any
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particular combination of allopatric forms (Table I). AMOVA indicated that thelargest amount of haplotypic variance (93·2%, P < 0·001) was distributed amonglakes with sympatric forms, whereas only 1·3% (P < 0·05) was distributed amongsympatric forms within lakes and 5·5% (P < 0·001) within sympatric forms(Table III).
DISCUSSION
P H Y L O G E N E T I C R E L AT I O N S H I P S O F SALVELINUS ALPINUSF RO M S I B E R I A
The present study is the first broad-scale investigation of Siberian S. alpinusmtDNA phylogeography. Haplotypes observed in this study clustered with the Arctic,Bering or Atlantic + Siberia + Acadia phylogenetic groups proposed by Brunneret al. (2001). The integrity of the Siberia group within the Atlantic + Siberia +Acadia supergroup implied by the scheme of Brunner et al. (2001), however, isnot supported. Several haplotypes of the Siberia group sensu Brunner et al. (2001)in Finland, the Taimyr region and on Spitsbergen (SIB1,2,3,5,9) are intermediatebetween other haplotypes found in Siberia (SIB4, 6, 8, 10–30), and the Atlantic grouphaplotypes (ATL1-18) (Figs 3 and 4). Thus, there is no evidence for the separationof the Atlantic and Siberia haplotypes into two distinctive groups equivalent to theBering and Arctic groups of Brunner et al. (2001). Furthermore, the discreteness ofthe Siberian and Atlantic groups was not clearly evident in the study of Shed’ko et al.(2007). Accordingly, all ASA S. alpinus from Eurasia can be considered as a singleEurasian group that includes the well-defined Atlantic subgroup and two putativesubgroups corresponding to the Siberia group of Brunner et al. (2001): Euro-Siberian(SIB1, 2, 3, 5, 9–Spitsbergen, Finland; Khatanga Bay and Pyasina Basins in Taimyr)and east Siberian [other haplotypes–Baikal and upper–middle Lena (Chaya, Vitim,Olekma) Basins in Transbaikalia; Pyasina, Khatanga and upper Khatanga Basinsin Taimyr; upper–middle Yana, Indigirka, and Lena (Aldan) Basins; Lena Delta,Olenek Basin] (Fig. 4). Although separated from the east Siberian subgroup in theHKY85 + I + �-based trees, haplotypes SIB26 and SIB29 (Fig. 3) are provisionallyplaced in this subgroup because of their positions in the haplotype network (Fig. 4), inthe trees based on other models and because of their distribution in inland continentalSiberia. The Eurasian group is genetically and geographically distinct from Acadiagroup S. alpinus from North America.
The causes of the discrepancies between the haplotypes found in one Acadian andthree Transbaikalian lakes in the study of Brunner et al. (2001) and in the presentstudy are unclear. Transbaikalian haplotypes observed in the present study fit into anexplainable phylogeographic pattern, being identical with haplotypes in neighbouringlakes. Haplotypes reported by Brunner et al. (2001) are inconsistent with this pattern,as they do not match with haplotypes in geographically neighbouring locations.
S P E C I E S B O U N DA R I E S A N D MTD NA I N T RO G R E S S I O N
Although this study does not primarily address taxonomic issues, particularlythe species-level boundary between S. alpinus and S. malma, the results link to
TABLE II. Haplotype diversity (h) and nucleotide diversity (π) in Salvelinus spp. populationsfrom Siberia grouped by locality, basin, region and phylogenetic group. Data on Salvelinusspp. from three localities in Taimyr (n = 8) studied by Brunner et al. (2001) are included*.
Numbers of localities correspond to those in Table I
Sample Locality, basin, region, Number of Haplotype Nucleotidenumber phylogenetic group N haplotypes diversity (h) diversity (π)
I Baikal 28 2 0·495 0·0010II Lena (Chaya) 56 1 0 0III Lena (Vitim) 155 10 0·810 0·0060IV Lena (Olekma) 154 5 0·617 0·0041V Lena (Aldan) 12 2 0·167 0·0007VI Lena (lower, delta) 16 2 0·400 0·0079VII Pyasina 10 3 0·622 0·0060VIII Khatanga 2 1 0 0IX Khatanga Bay 12 2 0·530 0·0073X Olenek 7 2 0·286 0·0056XI Yana (upper, middle) 19 3 0·205 0·0006XII Yana (delta) 8 1 0 0XIII Indigirka (upper,
middle)22 3 0·481 0·0061
XIV Indigirka (delta) 11 1 0 0XV Kolyma 5 1 0 0
Regionsa Transbaikalia (I–IV) 393 16 0·848 0·0066b Taimyr (VII–IX) 24 5 0·670 0·0070c Mountains of East
Siberia (V, XI, XIII,XV)
58 8 0·805 0·0075
d Coasts of East Siberia(VI, X, XII, XIV)
42 2 0·215 0·0042
Phylogenetic groupsA Eurasian group 475 26 0·892 0·0071B Bering group 42 2 0·215 0·0013
Total 517 28 0·903 0·0089
*Lake Lama (Pyasina Basin)–SIB7 (2), SIB 9 (2); Lake Ayan (Khatanga Basin)–SIB4 (2); Lake Arilakh(Khatanga Basin)–SIB5 (2).
this question. Haplotypes of the Bering group are typically found in northern S.malma in Kamchatka, Chukotka, Alaska, and north-western Canada (Brunner et al.,
P H Y L O G E O G R A P H Y O F S I B E R I A N S A LV E L I N U S A L P I N U S 385
TABLE III. Hierarchical analysis of molecular variance (AMOVA) of Arctic charr from Siberia
Degrees of Sum of % ofSource of variation freedom squares σ 2 variation
All Siberian lakesAmong regions 3 302·9 1·131 36·3Among basins within regions 11 346·2 0·969 31·1Within basin 501 510·1 1·018 32·6Total 515 1159·1 3·119 100·0
Siberian lakes with sympatric formsAmong lakes 22 577·4 1·703 93·2Among sympatric forms within lakes 29 6·5 0·024 1·3Within sympatric forms 302 30·2 0·100 5·5Total 353 614·1 1·826 100·0
2001; present study; Reist, unpublished data) and are introgressed in south AsianS. malma (Shed’ko et al., 2007). Haplotypes of other groups designated by Brunneret al. (2001) are typical for the S. alpinus complex. North American Arctic andBering haplotypes observed in this study and closely related haplotypes (Reist,unpublished data) align with one another, as expected for their respective speciesboundaries. In contrast, in several Siberian populations far to the west of the S.malma range, fish identifiable as S. alpinus bear haplotypes of the Bering group.These haplotypes have most likely introgressed into Siberian S. alpinus from PacificBasin S. malma as the result of earlier hybridizations. Introgression between thesetwo species has been found in previous studies (e.g. SINE’s, Hamada et al., 1998)and has been implied by allozyme data for North American populations (Reistet al., 1997).
Other explanations for the presence of Bering group haplotypes in S. alpinus arepossible. Shed’ko et al. (2007) postulated that Bering group haplotypes were initiallyrestricted to Alpinoid charr, were later transferred from them to northern S. malmaand subsequently became predominant in the gene pool of S. malma. Althoughcomplete substitution of native S. malma mtDNA seems unlikely, the appearanceof Bering group haplotypes in S. alpinus outside zones of sympatry may be viewedas evidence consistent with the Shed’ko et al. hypothesis.
G E O G R A P H I C A L D I S T R I B U T I O N O F H A P L OT Y P E SA N D D I S P E R S A L RO U T E S O F SALVELINUS ALPINUS I N S I B E R I A
Salvelinus alpinus exhibiting Eurasian group haplotypes are widely distributed incontinental Siberia from the Pyasina and Lake Baikal basins in the west to the Indi-girka Basin in the east and in Siberian Arctic coastal regions, from Khatanga Bayto the Lena Delta. Bering group haplotypes are common to the east along the coast,from Olenek Bay to the Indigirka Delta, and in the upper reaches of the Kolyma. Thegeographical ranges of the two groups overlap in the Olenek Bay and Lena Deltaregions. This suggests that charr colonized Siberia from the west and from the east.
First, Eurasian group S. alpinus from Europe dispersed along the northern Siberiancoasts in the mid-Pleistocene during one of the previous interglacial periods andascended to the upper reaches of Siberian rivers with climate cooling and theadvance of the northern ice sheets. Alternatively, but less likely, dispersal to the
continental regions of Siberia may have taken place through a system of largeperiglacial lakes (Grosswald, 1998). Colonization of Siberia by this group was rapidand proceeded from one diverse gene pool. Ancestral mtDNA polymorphism wasdifferentially retained in different basins and regions, thereby accounting for theobserved pattern of non or partly overlapping sets of haplotypes in them, for the lackof strict clustering of haplotypes by basin, and for the nearly equal amounts of geneticvariation distributed within and among basins, as well as among geographic regions.Glaciation in Siberia was dominated by separate mountain glaciations (Rozenbaum& Shpolyanskii, 2000) that allowed S. alpinus to survive in the low- or medium-elevation water bodies and to disperse into coldwater mountainous lakes as theclimate ameliorated. Isolated populations were formed in these lakes, and locallake-specific haplotypes appeared in some lakes, leading to increased local mtDNAdiversity in continental Siberia.
Second, in the coastal Siberian regions and on northern offshore islands, EurasianS. alpinus were presumably eliminated during the glacial epoch(s), and the west-ern Arctic coast and islands were re-colonized in the post-glacial period by Eurasiangroup S. alpinus from the west (Euro-Siberian subgroup) and from south-eastern con-tinental populations (east Siberian subgroup). Fish bearing Bering group haplotypesmay have re-colonized the eastern Siberian coast from the Pacific Basin. Pacific Basincolonists did not penetrate the upper reaches of Siberian rivers, except in the KolymaBasin, perhaps because these regions were already populated by Eurasian groupS. alpinus , or because interglacial climatic factors created warm lowland water bod-ies that precluded further upstream dispersal. Thus, in the northern Siberian coastalzone, a secondary contact between the Bering and the Eurasian groups, and betweenthe Euro-Siberian and the east Siberian subgroups has occurred.
One of the Bering group haplotypes (BER12) found in the Alaska and Yukonregions is identical, within the 485 bp fragment, with the most widespread haplotype(BER21) in the Asian Pacific Basin (Shed’ko et al., 2007). This indicates a closerelationship between and recent separation of Pacific Basin S. malma from the twocontinents.
P H Y L O G E O G R A P H I C PAT T E R N S I N T R A N S BA I K A L I A
Large mtDNA haplotype diversity was observed in Transbaikalia. The 16haplotypes observed in this area are divergent from one another and are not sharedwith coastal S. alpinus populations. By way of comparison, only three closely relatedhaplotypes are found in the S. alpinus populations of the European Alps (Brunneret al., 2001), an area of similar size, elevation, inland geographic position andriver basin segmentation. One haplotype (ATL1) is also present in coastal EuropeanS. alpinus, suggesting a recent colonization of the Alps by an anadromous form.Leaving aside the possibility of different mutational rates, the Alps may have beencolonized by S. alpinus later than the colonization of Transbaikalia (most probablyduring or after the last glaciation) and by more genetically homogenous and perhapsfewer immigrants.
The results of this study do not confirm the presence of a common haplotype(SIB4) in Transbaikalia and Taimyr (Brunner et al., 2001). Instead, haplotypesSIB8 and SIB10 were observed in common for Transbaikalia and the Yana Basinlakes in the Verkhoyanskii mountain range, which supports a northern origin of
P H Y L O G E O G R A P H Y O F S I B E R I A N S A LV E L I N U S A L P I N U S 387
Transbaikalian S. alpinus . The haplotype SIB10 likely represents an ancestralmitochondrial lineage of Transbaikalian S. alpinus , because of its position in thehaplotype network (Fig. 4), its wide geographical distribution in Transbaikalia andits occurrence beyond this region. This haplotype’s presence in S. alpinus fromthe Baikal Basin and the adjacent part of the Lena Basin indicates that S. alpinuspenetrated to the former from the latter and supports a previous discharge of LakeBaikal through the Lena (Mats et al., 2001). Another possibility is that S. alpinusdispersed to the Baikal Basin from the Lena Basin over the divide without a directconnection between the two basins.
Haplotypes are clearly segregated between the basins of the Lena tributaries inTransbaikalia (Chaya, Vitim and Olekma Rivers) and between some regions orsecond-order tributaries within these basins. This geographical pattern indicates thatS. alpinus dispersed throughout the Lena Basin following the formation of the presenthydrological configuration and that genetic divergences were associated mainly withseparation of populations in the tributaries. No migration between the tributariesappears to have occurred after populations of S. alpinus were established in theheadwaters, with the exception of occasional headwater captures. The results indicatean obvious case of capture of Lake Tokko [28] by the Chara sub-basin of the OlekmaBasin from the Khani Sub basin. The lake is connected to the former sub basin,but S. alpinus share a common haplotype with the latter. Lake Tokko is separatedfrom Lake Olongdo in the Khani Sub Basin by only 0·5 km of swampy marsh anddischarges in this direction, suggesting a previous connection between these lakes.Headwater captures promoting the transfer of Salvelinus spp. haplotypes betweenbasins, however, are probably rare in this area.
T H E O R I G I N O F S Y M PAT R I C F O R M S
The origins of sympatric S. alpinus forms have been addressed in several geneticstudies. Hindar et al. (1986) found much larger allozyme differentiation betweenallopatric Norwegian S. alpinus populations than between sympatric dwarf andnormal forms. The latter clustered closely together in two polymorphic populations,whereas in two others they clustered with Salvelinus spp. from allopatric populations.Multilocus minisatellite DNA analysis indicated greater genetic affinity of foursympatric morphs of S. alpinus in Thingvallavatn (Iceland) to each other, than ofany of them to S. alpinus from two allopatric populations, thus also suggestinga sympatric origin (Volpe & Ferguson, 1996). The analysis of microsatellitesdemonstrated that within each of four polymorphic Icelandic populations, sympatricforms of S. alpinus clustered together and shared unique alleles (Gislason et al.,1999). Analogous monophyletic grouping of sympatric populations was observedin six lakes in a wide-scale study of microsatellite variation of S. alpinus fromEurope (Wilson et al., 2004). The authors of both studies suggested the clusteringindicated, though did not unambiguously prove, a sympatric origin of forms. Infour other lakes surveyed by Wilson et al. (2004), sympatric forms showed closerrelationships to other populations than to each other, and thus their allopatric originwas strongly implied. In one of these lakes, the genetic distinctiveness of two morphsof S. alpinus was also demonstrated in an earlier single-locus minisatellite DNAanalysis (Hartley et al., 1995).
In Siberia, the study of allozyme variation in sympatric forms of S. alpinusin five Taimyr and Transbaikalian lakes revealed low or no divergence betweenthem (Osinov et al., 1996; Osinov, 2002). These data suggested an intralacustrineorigin of the forms through sympatric mechanisms (Osinov, 2002), but were notsufficient to reject the possibility of multiple invasions. Brunner et al. (2001) founddifferent mtDNA control region haplotypes and Radchenko (2003, 2004) observeddifferent restriction fragments of mtDNA ATPase6/ND4L and different mtDNAcytochrome b haplotypes in sympatric forms of S. alpinus from Lake Lama (Taimyr).Only one to two individuals of each form, however, were analysed in thesestudies.
The present work contributes additional data to the debate regarding speciationmodes in S. alpinus . The majority (n = 22) of polymorphic populations analysed inthis study were found in Transbaikalia. In this region, the closest genetic similarityis observed not between allopatric populations of analogous (dwarf, small, orlarge) forms of S. alpinus , but between intralacustrine forms. Thus, these threeforms are not monophyletic lineages that independently dispersed throughout theregion, but rather are polyphyletic and repeatedly evolved de novo in differentlakes. The present results support the sympatric origin of S. alpinus forms in sixTransbaikalian lakes, where sympatric forms share lake-specific haplotypes. BecauseTransbaikalia has been thoroughly screened for charr lakes, and this study includedall known Transbaikalian populations, it is unlikely that these haplotypes will befound elsewhere in this region, and it can be assumed that they appeared in each ofthe respective lakes in the common ancestors of sympatric forms. Although sharedunique haplotypes in sympatric forms could also be due to introgression, it is unlikelythat introgression led to complete (or nearly complete) substitution of haplotypesin one of the forms in several lakes. In the remaining 16 lakes, sympatric formsshare haplotypes not only with each other, but with S. alpinus in adjacent lakes.Accordingly, their immigration from nearby areas cannot be precluded by mtDNAdata. Genetic or other evidence, however, fails to support multiple invasions to anyof these lakes from neighbouring lakes. Thus, intralacustrine differentiation of formsfollowing the sympatric speciation model seems to be the most probable evolutionaryscenario in all or the majority of these polymorphic Transbaikalian populations ofS. alpinus .
The possible exceptions to this are closely interconnected lakes (Bol’shoe Leprindo[23], Maloe Leprindo [22] and Gol’tsovoe [21]; Kiryalta-3 [25] and Kiryalta-4 [26]).Outside Transbaikalia, a polymorphic population was observed in Lake Tunernde [35]in the Aldan Basin. The sympatric forms of S. alpinus shared a unique haplotype,thus also implying a sympatric mode of evolution in these instances. This region,however, is poorly studied and it is not possible to draw firm conclusions aboutdifferentiation modes between forms of S. alpinus from available data.
Sympatric forms originating by multiple invasions, if any, are more likely to existin northern Siberian regions in the zones of secondary contact of Bering and Eurasiangroups, and subgroups of the latter. Control region haplotypes observed by Brunneret al. (2001) in S. alpinus complex forms from Lake Lama belong to Eurosiberian andeast Siberian subgroups (SIB9 and SIB7, respectively). If correspondence betweenforms and haplotypes is confirmed in larger samples, this can be considered asevidence of allopatric origin of these forms (Romanov, 2003).
P H Y L O G E O G R A P H Y O F S I B E R I A N S A LV E L I N U S A L P I N U S 389
We thank colleagues for providing samples of charr: M. Yu. Pichugin (Moscow StateUniversity) – from Lake Kungasalakh and Novaya River, O. L. Makarova, A. B. Babenko(Institute of Ecology and Evolution, RAS, Moscow) and D. N. Krasavin (meteorologicalstation Vostochnaya, Sakha-Yakutia) – from lakes Kobyuma-1, 1a, Krugloe and Rys’, P. M.Tychkin – from Lake Usu; Irene Gregory (University of Ottawa) for samples from Karlukand Fraser Lakes; Fred Kircheis (Maine Fisheries) from Floods Pond. We are grateful toS. G. Afanas’ev, N. V. Alekseyeva, A. S. Alekseyev, B. E. Bogdanov, V. V. Buldygerov,I. B. Knizhin, A. G. Osinov, M. Yu. Pichugin, V. V. Pulyarov, F. N. Shkil’, A. A. Sokolov(deceased), A. I. Vokin, A. L. Yur’ev, J. A. Babaluk and J. Hammar for field assistance, andto two anonymous reviewers for valuable comments on the manuscript. Financial supportfor this study was provided by a NATO collaborative linkage grant to M.P., S.S.A. andJ.D.R., by grants from the Russian Foundation for Basic Research (projects 08-04-00061,05-04-97262), from the Programme of Fundamental Studies of the Presidium of the RussianAcademy of Sciences, Biodiversity and the Dynamics of Gene Pools [projects entitled Thestudy of the mechanisms of intraspecific variability (morphotypes, ecological forms) as afactor of the formation of biodiversity (5.4.3) and Genetic processes in population systems ofanimals, plants and humans: factors of stability and evolution) and from the President of theRussian Federation, project MK-5555.2008.4. Completion of the study was conducted underthe Canadian International Polar Year project entitled Climate variability and change effectson chars in the Arctic funded by Indian and Northern Affairs Canada.
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