The Role of Nuclear Genes in Intraspecific Evolutionary Inference: Genealogy of the transferrin Gene in the Brown Trout

Agostinho Antunes*{dagger}{ddagger},Go, Alan R. Templeton§, René Guyomard{dagger} and Paulo Alexandrino*{dagger}

*Centre de Investigação em Biodiversidade e Recursos Genéticos (CIBIO/UP), Campus Agrário de Vairão, Vairão, Portugal;
{dagger}Departamento de Zoologia-Antropologia, Faculdade de Ciências, Universidade do Porto, Praça Gomes Teixeira, Porto, Portugal;
{ddagger}Laboratoire de Génétique des Poissons, INRA, Jouy-en-Josas, France;
§Department of Biology, Washington University, St. Louis


    Abstract
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Technical and biological hurdles have precluded the retrieval of nuclear gene genealogies within most species. Among these obstacles, the possibility of intragenic recombination is one of the most demanding challenges. We studied the utility of nuclear genes for intraspecific evolutionary inferences by selecting a nuclear gene that exhibits patterns of considerable geographic differentiation in the brown trout (Salmo trutta) species complex. Haplotype variation from a nucleotide sequence of ~3.7 kb encompassing a portion of the transferrin (TF) gene was surveyed in 31 brown trout individuals collected across the native Eurasian range. Statistically significant recombination and gene conversion events were detected. However, we showed that the substantial cladistic structure was not disrupted by recombination or gene conversion events and the additional structure was estimated to have emerged after those events. Because loci with unusually high levels of variation might indicate the presence of selection, we tested the hypothesis of neutrality and found some evidence for directional selection. The strong geographic signal observed in the TF genealogy, coupled with the current spatial distribution of electromorphs, gave us the ability to draw empirical phylogeographic inferences. We delineated the composition of current brown trout populations on the basis of 3,625 individuals electrophoretically scored for the TF locus. We hypothesized scenarios of historical radiation and dispersal events, thus providing new insights refining previous allozyme and mtDNA inferences. We infer that the most ancestral brown trout populations inhabited tributaries from the Black, Caspian, and Aral Sea drainages. An early radiation of the species occurred throughout the Mediterranean, followed by independent dispersal events from the Adriatic to the southernmost Iberian Atlantic and, more recently, a rapid expansion throughout most of the Atlantic drainages.


    Introduction
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
The study of the principles and processes governing the geographic distribution of genealogical lineages, phylogeography, has often focused on the mitochondrial (mt) genome (Avise 1998Citation ). Yet, the matrilineal pathways of ancestry registered by this molecule represent only a small portion of the total historical record of a sexual organismal pedigree (Avise and Wollenberg 1997Citation ). Much of the remainder of that history should be inferable from autosomal gene trees. However, as a result of its high complexity, few attempts have been made to estimate nuclear gene genealogies in a phylogeographic framework outside of humans (e.g., Palumbi and Baker 1994Citation ; Hare and Avise 1998Citation ). Documenting the historical incidence of intragenic recombination within a species is one of the most difficult challenges of nuclear genealogies (Avise 2000, pp. 90–102Citation ). However, recent methodologies have been proposed to overcome this difficulty so as to provide a tool to detect statistically significant recombination events (Crandall and Templeton 1999Citation ; Templeton et al. 2000aCitation ) and reveal the evolutionary gene structure not disrupted by recombination (Templeton et al. 2000bCitation ).

Presently, many allozyme and protein loci have been subject to DNA sequencing and offer the opportunity to understand the genealogy of classical nuclear markers. Such an understanding could be especially useful where large data sets have been generated previously for such markers, allowing the choice of a locus exhibiting significant differentiation among populations at large and small geographic scales. That is the case of the transferrin (TF) locus, which is polymorphic in many vertebrate species, including fish (Kirpichnikov 1981, pp. 162–168Citation ). The locus codes an iron-binding protein found in vertebrate blood serum and interstitial spaces (Loehr 1989Citation , pp. 241–243). Among salmonids, the locus has frequently been used to differentiate populations (e.g., Payne 1974Citation ; Krieg and Guyomard 1985Citation ; Van Doornik, Milner, and Winans 1996Citation ) and, more recently, has received attention in studies of molecular adaptation (Ford, Thornton, and Park 1999Citation ; Ford 2000Citation , 2001Citation ).

In this context, the brown trout (Salmo trutta L.) is an appropriate organism to study the utility of nuclear gene genealogies for evolutionary inferences. This species exhibits a strong genetic structuring and is considered a polytypic species with complex patterns of phenotypic diversity and life history variation, including anadromous, fluviatile, and lacustrine modes of life (Behnke 1972Citation ). Over the last two decades, geographic patterns of genetic differentiation were often observed, using both allozymes (e.g., Ryman, Allendorf, and Ståhl 1979Citation ; Guyomard 1989Citation ; Giuffra, Guyomard, and Forneris 1996Citation ; Bouza et al. 1999Citation ) and mtDNA (e.g., Bernatchez, Guyomard, and Bonhomme 1992Citation ; Giuffra, Bernatchez, and Guyomard 1994Citation ; Bernatchez and Osinov 1995Citation ; Apostolidis et al. 1997Citation ; Antunes et al. 2001Citation ). The sequencing of the mtDNA control region delineated five major evolutionary lineages (Danubian [DA], Adriatic [AD], marbled trout [MA] found in some Adriatic drainages, Mediterranean [ME], and Atlantic [AT]; Bernatchez, Guyomard, and Bonhomme 1992Citation ; Bernatchez 2001Citation ) showing a strong distribution according to major drainage basins (fig. 1 ). The distribution of allozyme variants is mostly congruent with these lineages (e.g., García-Marín, Utter, and Pla 1999Citation ).



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Fig. 1.—(A) Schematic representation showing the geographic distribution of mtDNA lineages according to major drainage basins: Atlantic (AT), Mediterranean (ME), Adriatic (AD), marbled trout (MA; found in some Adriatic drainages), and Danube (DA) which extends further to other Black Sea drainages, and Caspian and Aral Sea drainage basins (Bernatchez and Osinov 1995Citation ) (adapted from Bernatchez, Guyomard, and Bonhomme 1992Citation ). (B) Maximum-parsimony consensus tree for the five major brown trout mtDNA evolutionary lineages (adapted from Bernatchez 2001Citation ). The relationships among major lineages and their bootstrap values (as a percentage) resulted from the combined information of three studies (Bernatchez, Guyomard, and Bonhomme 1992Citation ; Giuffra, Bernatchez, and Guyomard 1994Citation ; Bernatchez and Osinov 1995Citation ) based on sequences (1.25 kb) from both the mtDNA control region and coding genes (cytochrome b and ATPase subunit VI). The numbers in parentheses refer to the number of apomorphies unique to each lineage. Branch lengths were scaled using the number of mutations (the shortest corresponding to a single mutation). To simplify the visualization of the main mtDNA lineages, haplotype designations have been removed

 
Considering the TF locus alone, substantial geographic patterns of electrophoretic variants were described in the brown trout (e.g., Guyomard 1989Citation ; Giuffra, Guyomard, and Forneris 1996Citation ; Largiadèr and Scholl 1996aCitation ) (fig. 2 ). The electromorph TF*100 is generally fixed for Atlantic populations and domesticated hatchery strains. TF*102 is often fixed for the Mediterranean brown trout, TF*75 for the marbled trout (S. trutta marmoratus), and TF*78 for the Italian carpione (S. trutta carpio). Other electromorphs were observed in moderate to high frequencies, such as TF*95 in southwestern Atlantic populations from Portugal (Antunes, Alexandrino, and Ferrand 1999Citation ) and TF*80 in Corsican populations (Krieg and Guyomard 1985Citation ; Presa et al. 1994Citation ; Berrebi 1995Citation ). The Atlantic salmon (S. salar) presents both the TF*80 and *102 electromorphs (Giuffra, Guyomard, and Forneris 1996Citation ). The electrophoretic analysis of allozyme or protein variation, however, shows an inherent limitation for some phylogenetic purposes in that the historical relationships of alleles remained unresolved.



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Fig. 2.—Geographic locations of the brown trout specimens analyzed. Numbers correspond to individual codes from table 1 . The shaded area of the map represents the current distribution of the species complex (adapted from Guyomard 1989Citation ; Elliott 1994Citation , pp. 9–10). The dark-shaded area indicates the presence of the anadromous form. The geographic distribution of the TF electromorphs is also shown. The electromorph TF*101 was reported for the first time in this study (see Material and Methods for details). The sizes of the electromorph boxes are intended to give a relative idea of their population frequencies. A detailed description of their distribution and frequencies is provided in the Supplementary Materials

 
In this study, we analyze sequence variation from the TF gene in the S. trutta species complex over most of its native Eurasian range, with samples representing the major electrophoretic variants. We intend to (1) document the cladistic structure of the gene and its utility for evolutionary inferences, (2) examine the roles of both recombination-gene conversion and mutation, (3) test the hypothesis that patterns of variation among the TF locus can be explained by the neutral model (Kimura 1968Citation ), and (4) obtain a phylogeographic portrait for the brown trout by overlaying the TF genealogy with the geographic distribution of the different electromorphs.


    Material and Methods
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Molecular Study of TF Electromorphs
The current geographic distribution of TF electromorphs was assessed by combining previously published data (Berrebi 1995Citation ; Giuffra, Guyomard, and Forneris 1996Citation ; Berrebi et al. 2000Citation ) with new electrophoretic screenings from several geographic areas (31 European populations corresponding to 661 individuals; see Supplementary Material at MBE website: www.molbiolevol.org). For this study, electromorphs were typed by agarose gel electrophoresis (AGE) (Antunes, Ferrand, and Alexandrino 2000Citation ) or isoelectric focusing (IEF) (Antunes, Alexandrino, and Ferrand 1999Citation ) (or both). A previously unknown electromorph (TF*101) was detected in Spanish populations from the Mediterranean drainage, as confirmed by the side-by-side running of reference samples (Largiadèr and Scholl 1996aCitation ), both in AGE and in IEF separation systems. A total of 31 brown trout samples exhibiting electromorphs TF*75, *78, *80, *95, *100, *101, and *102 (table 1 ; fig. 2 ) was selected for sequence analysis. However, for eight individuals coming from tributaries of the Black, Caspian, and Aral Sea drainages, electromorph information was not available. We avoided samples coming from populations with genetic evidence of introgressive hybridization resulting from stocking activity or contemporary human-induced habitat disturbance. Two Atlantic salmon (S. salar) specimens homozygous for TF*80 were also examined and used as an outgroup in the phylogenetic analysis.


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Table 1 Geographic Origins, Subspecific Designation, Electromorphs, Haplotypes, and a.a. Sequences of the Individuals Analyzed

 
Sequencing was performed on diploid TF genotypes for the selected samples (table 1 ). The gene is well conserved among vertebrates and organized into 17 exons separated by 16 introns (Schaeffer et al. 1987Citation ; Mikawa, Hirono, and Aoki 1996Citation ; Ford, Thornton, and Park 1999Citation ). The segments used in this study were selected after a preliminary analysis of almost the entire TF gene (around 8 kb sequenced) from a brown trout and an Atlantic salmon, and a preliminary survey of variable regions among several brown trout specimens. We avoided portions that exhibited extensive length polymorphism caused by tandem repeats or indels because such variation makes direct sequencing difficult or impossible. For each individual, exons 2, 4, 6, 7, and 13 and introns 1, 3, 5, 6, 12, and 13 were completely sequenced. Exons 1, 3, 5, 8, 10, 12, and 14 and introns 2, 4, 7, and 9 were partially sequenced (fig. 3 ). Genomic DNA was extracted from blood or muscle tissue following Sambrook, Fritsch, and Maniatis (1989Citation , pp. 9.16–9.19). Sequences were amplified via the polymerase chain reaction (PCR). Primers were modified from those of the coho salmon (Oncorhynchus tshawytscha; Ford, Thornton, and Park 1999Citation ), with slight adjustments based on published cDNA in brown trout and Atlantic salmon (Kvingedal, Aleström, and Rørvik 1993Citation ; Lee et al. 1998Citation ): TF-ex1-F 5'-CATGAAACTGCTTCTCCTCTC-3', TF-ex3-R 5'-CCTCACCATAGTCCTCTGCAAT-3', TF-ex3-F 5'-GCCTCACTAACTACGGCCTGCA-3', TF-ex5-R 5'-TGGAAGGCCCCAGCATAGTCAT-3', TF-ex5-F 5'-AGGTCTCACAAGGAGCCCTA-'3, TF-ex7-R 5'-TTGACGGCCACCAGTTTGTTG-'3, TF-ex7-F 5'-CGCAAGGACCCCGAACTGGC-3', TF-ex10-R 5'-ATACTGCTCCACCATGACAGGG-3', TF-ex12-F 5'-TACCCATGGGTCTCATCCACAA-3', and TF-ex14-R 5'-CATCAGTGCTCTCTGGTACAAT-3'. Two new primers were also designed: TF-ex8-R 5'-GGCAGCTGTACTAGTTTCTGAG-3' and TF-ex9-F 5'-TTCAGCGCAGGCCACAGGTG-3' (see fig. 3 ). PCR fragments were generated using the following profile: 5 min of denaturation at 94°C; 37 cycles of denaturation for 50 s at 92°C and annealing for 50 s at 57–60°C (depending on the primer set); and extension for 70 s at 72°C. The last cycle was followed by an additional 5 min at 72°C. Sequences were generated with BigDye Terminator Cycle Sequencing protocols on an ABI-310 automated sequencer (PE Applied Biosystems) and checked by hand using the Sequence Navigator® software (version 1.0.1; Applied Biosystems, Inc.). Alignments were performed using SeqApp (Gilbert 1996Citation ).



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Fig. 3.—Schematic diagram of the TF gene regions surveyed. The black boxes and lines represent the exon (exn) and intron (inn) domains, respectively. Primer sets for each gene segment are indicated below the diagram (the TF-ex9-F primer, although not represented, was used in conjunction with TF-ex10-R). Gray boxes identify the physical location of the three gene regions with recombinant sites: I (165–779 bp), II (1759–1928 bp), and III (2933–3649 bp). Variable sites involved in crossover events are identified by gray bars

 
Haplotypes were determined by the haplotype-subtraction algorithm of Clark (1990)Citation . Confirmatory analyses were performed by molecular cloning (InsT/A Clone PCR Product Cloning Kit). Not all the haplotypes were observed directly, so some are not known with certainty. Sequence comparisons, measures of variability, and translation of exon sequences into amino acids (a.a.) were performed using MEGA (version 2.1; Kumar et al. 2001Citation ).

Estimation and Testing of Recombination-Gene Conversion
Detection of recombination-gene conversion events was performed using the CT algorithm of Crandall and Templeton (1999)Citation with additions given in Templeton et al. (2000a)Citation . The algorithm starts with the estimation of a haplotype "tree" under the null hypothesis of no recombination or gene conversion. The quotation marks placed around the word tree are to emphasize that this tree may not be an accurate reflection of the evolutionary history if the null hypothesis is rejected. From the checked robustness of the different tree topologies in Templeton et al. (2000a)Citation , we chose the statistical parsimony (SP) method for the tree estimation. SP (Templeton, Crandall, and Sing 1992Citation ; Crandall 1994Citation ; Crandall and Templeton 1996Citation ) favors parsimonious solutions that avoid placement of homoplasies on short branches that lie within the "limit of parsimony" (Templeton, Crandall, and Sing 1992Citation ). Maximum-parsimony trees were generated using PAUP (version 4.0b2a; Swofford 1998Citation ). Comparison of the adjusted character distances with the corresponding patristic distances allowed the elimination of that maximum-parsimony trees that violated the limits of parsimony. The generated trees were also compared with the correspondent networks estimated in TCS (version 1.13; Clement, Posada, and Crandall 2000Citation ).

After the tree estimation, the null hypothesis of no recombination was tested by means of associations between the positions of apparent homoplasies in the tree and the order of physical positions in the DNA sequence. This association is used in the CT algorithm to identify putative recombinants, tracing pathways through the tree, and to match the homoplasies clustered on a branch with their occurrences elsewhere in the tree. The algorithm also allows the identification of crossover intervals and candidate parental types. To identify statistically significant recombinants, a runs test based on a hypergeometric distribution was used. A program in MATHEMATICA (Wolfram 1996Citation ) was written to perform those tests (Templeton et al. 2000aCitation ).

The CT hypergeometric test is based on the ideal expectation of two runs under recombination: {alpha}-matched homoplasious sites from one parental type defining a run on one end of the physical sequence, followed by a run of ß sites (i.e., the mutations on the pathway interconnecting the matched homoplasies) from the other parental type on the other end of the sequence. Gene conversion is another genetic mechanism for placing a physical cluster of nucleotide states on a new haplotype background. It can place a small run of variable sites from one haplotype into the middle of a second one, thereby yielding three runs by physical location. It can also result in only two runs, but such cases are indistinguishable from recombination. After the recombination tests were performed, an additional runs test was carried out to detect gene conversion events that have resulted in three or more runs. To test gene conversion with more than two runs, {alpha}-apparent homoplasies on a branch are matched with another region of the tree with ß-other mutations lying on the pathway between the two tree regions (Crandall and Templeton 1999Citation ). The {alpha} + ß mutations are then ordered by physical position, and the number of runs, say {delta}, of {alpha} and ß mutations in the physically ordered sequence is recorded. Under the null hypothesis of no recombination-gene conversion, the probability for the number of runs, according to Mood and Graybill (1963, pp. 409–412)Citation , is given by:


Because gene conversion is tested only after recombination (d = 2), equations (1) are used to calculate the probability of {delta} = 2 under the null hypothesis, and then equations (1) are divided by [1 - Prob({delta} = 2)] to obtain the conditional distribution of runs, given that d >= 3, the only condition under which this gene conversion test is used. For more details, see the work of Crandall and Templeton (1999)Citation and Templeton et al. (2000a)Citation .

Inferring the Cladistic Structure in the Presence of Recombination-Gene Conversion
In this study we have inferred the cladistic structure of the TF gene using primarily an alternative and novel method recently proposed by Templeton et al. (2000b)Citation for removing the effects of recombination and estimating the cladistic structure in a DNA region that has experienced recombination. This approach has a broader range of applicability than the method of subdividing a gene into smaller regions that show little or no internal recombination (Templeton et al. 2000bCitation ). The analysis starts by removing all inferred recombination events from the SP tree estimated for the entire DNA region. This means that the recombinant haplotypes identified by the CT algorithm are removed from the SP tree because homoplasies do not represent actual mutational events. All haplotypes and branches that are derived from such recombinant haplotypes by subsequent events are also removed. The remaining portion of the SP tree after this removal, the peeled tree, ideally reflects the component of haplotype diversity that has not been affected by recombination during the coalescence of this DNA region (Templeton et al. 2000bCitation ). However, additional cladistic structure could have arisen in haplotype lineages derived from recombinant haplotypes. This postrecombinational cladistic structure is estimated by those subsets of the SP tree that consist of branches and haplotypes derived from each of the original recombinants by subsequent mutational events.

Tests of Neutrality
We tested the fit to the neutral expectations of nonsynonymous and synonymous segregating sites within brown trout using a contingency table (G-test; Templeton 1996Citation ). From the inferred cladistic structure (peeled tree and recombinant clades), two tree topological mutational categories were used: tip and interior branches. The topological contrast of tip versus interior for the most part corresponds to a contrast of young versus old, thus yielding heterogeneity when selection occurs (i.e., tips are new and unproven, whereas interiors are older and have left descendants and thus have proven evolutionary success). Further, six functional mutational categories were considered: silent and replacement substitutions in hypothesized positively selected sites (PSSs) in salmonids (Ford 2001Citation ), silent and replacement substitutions flanking PSSs (±3 codons), and silent and replacement substitutions in other sites. Two-by-two contingency tables were then analyzed using Fisher's exact test (FET) in STATISTICA (StatSoft 1993Citation ). An exact permutational test on the entire two-by-six matrix was performed using CHIPERM (version 1.0; Posada 1998Citation ).


    Results
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Electrophoretic Screening, Sequence Variation, and Haplotype Determination
Individuals (3,625) were electrophoretically scored for the TF locus (see Supplementary Material for details). Figure 2 depicts the observed geographic distribution of the TF electromorphs.

Noncontiguous fragments of 3,696 bp (including indels) representing ~44% of the full length of the TF gene (as compared with the homologous gene in medaka fish [Oryzias latipes]; Mikawa, Hirono, and Aoki 1996Citation ) were sequenced in 31 brown trout and two Atlantic salmon individuals (table 1 ; fig. 2 ), describing a total of ~122 kb analyzed (GenBank accession numbers AF488833AF489096). The aligned sequence data revealed 173 variable sites (excluding intraspecific variable sites within the Atlantic salmon), 113 of which varied within the brown trout. Of those, 106 (including 13 indels) were used for haplotype reconstruction and then for phylogenetic inferences (table 2 ). The nucleotide divergence was slightly less for codon sites (table 3 ).


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Table 2 Sequence Variants Used to Define Haplotypes in the Brown Trout TF Gene

 

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Table 3 Observed Number of Transition/Transversion Pairs of Nucleotides and Estimation of the Number of Nucleotide Substitutions per Site (p-distance in codon and overall positions) Between the DNA Sequences for the Brown Trout TF Gene (excluding gaps)

 
In the haplotype determination, five nucleotide positions (101, 579, 2910, 3059, and 3407), often heterozygous in most diploid individuals sequenced, were excluded because they lead to the premature termination of the cascade of inference for the haplotype-subtraction algorithm of Clark (1990)Citation . Also, the length variation at a dinucleotide repeat (positions 3210–3217) was excluded from haplotype determination because this variation does not fall under the same evolutionary models as the other variable sites. For the genotypically heterozygous individuals 2, 3, 4, 6, 10, and 26, the unambiguous inference of two haplotypes was not possible. Therefore, two possible sets of haplotypes were estimated (table 1 ). This haplotype ambiguity often resulted in loops of ambiguity on the estimated SP tree (mainly in tip branches; fig. 5 ) and contributed to an overestimation of the total number of different haplotypes (n = 48). These ambiguities could be resolved in the future by applying a population frequency criterion proposed by Crandall and Templeton (1993)Citation or via molecular haplotyping. For the present, the ambiguities were retained, represented as dashed lines for links that were nearly equally probable.



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Fig. 5.—The total haplotype network estimated under the null hypothesis of no recombination or gene conversation. Small circles indicate nodes in the tree that represent intermediate haplotype states not found in the sample. Each line (solid or dashed) represents a single mutational event. The site involved in the mutation is indicated near the line by a small boldface number, with the numbers corresponding to the variable site numbers given in table 2 . The sites involved in crossover events are boxed (homoplasies on a branch, represented by boxed gray sites, are matched to another region of the tree, represented by black-boxed sites; see also figs. 6 and 7 ). Dashed lines indicate where loops or alternatives create ambiguity in the topology of the tree. Nodes that define the three major termini are indicated by an oval containing T-i, where i can be 1, 2, or 3. Other nodes involved in recombination events are indicated by the boldface lowercase letters a–e. Haplotypes observed more than once are shaded gray. Haplotypes observed more than once, but resulting from an overestimation haplotype determination (see Results), are boxed. The black salmonid fish indicates the connection with the outgroup (S. salar) and hence indicates the rooting of the tree. Electromorph designation was labeled in the tree to help visualize the relationship between haplogroups and electromorphs. The haplotypes of individuals from tributaries of the Black, Caspian, and Aral Sea drainages were arbitrarily designated as TF-BCA because no electromorph information was available

 
Generally, the individuals scored as phenotypically homozygous for a TF electromorph were genotypically homozygous or heterozygous for a sequence differing by few nucleotides (<=10). Yet, individuals 7, 13, 22, and 23 were heterozygous for alleles differing by a large number of nucleotides (23–40). Although the electromorph was not available for individual 7, the other three individuals exhibited one haplotype corresponding to the electromorph for which they were phenotypically scored as homozygous and another haplotype corresponding to a distinct electromorph.

The a.a. sequences inferred from the translation of exons revealed 356 sites representing ~52% of the total length of the TF protein in salmonids (Lee et al. 1998Citation ). The aligned sequence data for all individuals revealed 13 different sequences with 27 variable positions, 15 of them varying within brown trout (fig. 4 ). With the exception of two sites, all brown trout a.a. polymorphisms were biallelic (positions 312 and 320 had three alleles). The relation between electromorphs and their corresponding partial a.a. sequences revealed that some electromorphs have identical partial sequences (TF*78 and *80 = TFaa78.80; TF*101 and *102 = TFaa102), whereas TF*100 exhibited two different sequences (TFaa100-1 and TFaa100-2). TF*80, shared by Atlantic salmon and brown trout, exhibited very distinct a.a. sequences. The a.a. divergence among brown trout varied between 1.4% and 1.6%. The nucleotide divergence among haplotypes varied between 0.1% and 1.1%. Overall, we detect a strong correlation between the nucleotide and a.a. sequence and the correspondent electromorph.



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Fig. 4.—Alignment of inferred partial TF protein sequences in the brown trout. Points indicate the identity of a.a. residues with one of the haplotypes of the outgroup (S. salar) (see table 1 ). Exon segments (exn) are indicated on the top of the alignment. Exons 3 and 5 are represented by two noncontiguous segments jointed at positions 72 and 168, respectively (indicated by "{vee}"). Residues of a.a. thought to be positively selected in salmonids (Ford 2001Citation ) are shaded

 
Phylogenetic Inference Under the Null Hypothesis of No Recombination-Gene Conversion
The SP tree estimated under the null hypothesis of no recombination and gene conversion is presented in figure 5 .

Detection of Statistically Significant Recombination-Gene Conversion Events
Two recombination and five gene conversion events were significant at the 5% level and were identified by applying the algorithm of Crandall and Templeton (1999)Citation to the null hypothesis tree. Figure 6 shows the tail probability from the hypergeometric test of the null hypothesis of no recombination and gene conversion and the spanned variable sites involved in the crossover event. Most of the variable sites were nucleotide substitutions, but a few involved insertions or deletions. Physical locations of recombinant sites comprise three regions in the gene: (I), a portion of exon 2 and intron 3, and intron 2; (II), a portion of intron 5; and (III), a portion of intron 9, and exons 10, 13, and 14 (fig. 3 ). Recombination-gene conversion events were identified in regions (I), (II), and (III) at the following frequencies: 1, 3, and 2, respectively.



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Fig. 6.—The estimated recombinant clades. Solid and dashed arrows represent recombination and gene conversion events, respectively. The P value near the arrow corresponds to the tail probability from the hypergeometric test of the null hypothesis of no recombination and gene conversion, and the spanned variable sites involved in the crossover event are indicated in parentheses. The layout of the network is the same as that given in figure 5

 
Estimation of the TF Gene Genealogy
Figure 7 presents the cladistic structure that remains after the removal of all significant recombination or gene conversion events, the homoplasies attributable to them, and any additional structure that has evolved from the original recombinant-converted haplotypes. Figure 6 shows the two recombination and five gene conversion events, along with the cladistic structure estimated to have arisen after the event. Because recombination-gene conversion was not concentrated in a single hot spot region (fig. 3 ), subdivision of the DNA sequence studied in smaller segments that showed little or no internal recombination would have discarded more than 40% of our data set. As a result, the SP trees of the subdivided DNA region, the 5' region (1–164 bp), the internal region (780–1758 bp), and the 3' region (1929–2932 bp) were not as informative as the peeled tree and recombinant clades estimated under the methodology proposed by Templeton et al. (2000b)Citation (data not shown). The few topological discrepancies between the subregional trees and the peeled tree could be explained by a substantial reduction of the informative variable sites within each subregion.



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Fig. 7.—The estimated haplotype tree after all recombinant and gene conversion events have been removed. The layout of the network is the same as that given in figure 5

 
Tests of Neutrality
Contingency tables were constructed by counting the number of mutational events in the various categories and the various types of branches for the postrecombinant clades and the peeled tree (figs. 6 and 7 ). From the 29 positively selected a.a. sites in salmonid TF defined by Ford (2001)Citation , 20 were part of our data set (fig. 4 ). The contingency tables and test results are shown in tables 4 and 5 . For the different two-by-two tests, none of the results were significant at the 5% level. However, the two-by-six test was shown to be highly significant (P < 0.001).


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Table 4 Topological and Functional Mutational Categories Used for the Contingency Analysis

 

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Table 5 Contingency Analysis of the Different Mutational Categories Tested

 

    Discussion
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Electrophoretic Versus Molecular Variation
Considerable molecular differentiation was found between and within the distinct TF electromorphs analyzed. Many different haplotypes were lumped into a single electromorph class. Substitutional variants within an electromorph generally did not reveal additional a.a. changes. Yet, for the electromorph *100 we observed two protein sequences differing by one a.a. Alternatively, examples of convergent mobility were identified, e.g., the electromorph TF*80 in Atlantic salmon and brown trout differed by 18 a.a. This provides empirical evidence for the limitation of gel electrophoresis as a detector of genetic variation where comparisons are made at the generic level (Ramshaw, Coyne, and Lewontin 1979Citation ; Barbadilla, King, and Lewontin 1996Citation ). Furthermore, it demonstrates the danger of inferring ancestral relationships of electromorphs solely on the basis of shared mobility among taxa.

On the other hand, we found cases where a.a. differences between electromorphs were not detected in the partial nucleotide sequences analyzed. The distinct electromorph TF*101 was not differentiated at the sequence level from TF*102 because both had identical a.a. sequences and shared nucleotide haplotypes in the portion of the gene sequenced. Electromorphs TF*78 and *80 also exhibited identical a.a. sequences; however, nucleotide sequences were different between electromorphs. In both cases, the detected electrophoretic variation presumably results from mutations in other coding regions of the gene.

TF Gene Genealogy
One traditional method for estimating the cladistic structure in a DNA region subject to recombination involves removing the impact of recombination by splitting the DNA region into subsegments with little or no recombination (e.g., Templeton and Sing 1993Citation ). However, the method would be difficult to implement under uniform recombination unless recombination was either rare or concentrated into hot spots (or both). Here, we implemented the method of Templeton et al. (2000b)Citation and identified three distinct areas of recombination and no single hot spot.

In spite of the observed recombination-gene conversion events in the portion of the TF gene analyzed, substantial cladistic structure was discriminated. The peeled tree (fig. 7 ) provides an estimate of the cladistic structure contained within the 3.7-kb region sequenced that has not been altered by any detected recombination or gene conversion events. This peeled tree contains the branches leading to the three major termini (T-1, T-2, and T-3) that were defined primarily by sites 5' to the recombinational region I (fig. 3 ). Moreover, the fact that the branches among the three major termini resulted in part from rare genomic changes (RGCs; Rokas and Holland 2000Citation ) implies that this evolutionary structure is quite old.

Substantial cladistic structure has also arisen after recombination-gene conversion events (fig. 6 ). Interestingly, those events provide evidence for secondary intergradation between distinct subclades. Some of these occur currently in the same geographic region, such as the TF95 and TF100 (Atlantic drainage), and the TF102 and TF78.80 (Mediterranean drainage) subclades. Gene conversion between subclades TF-BCA x TF102 and TF-BCA x TF75 currently occupying distinct geographic areas (Black, Caspian, and Aral Sea, as opposed to Adriatic and Mediterranean drainages), could represent a signature of past intergradations between those subclades. Additional evidence for secondary contact and introgressive hybridization is represented by the R1 and R2 haplotypes in individual 7, from the upper Danube. These haplotypes are related to the TF100 subclade, but they evolved by gene conversion and recombination events with other subclades (fig. 6 ). This result is consistent with previous studies, which showed that some unstocked populations from the Danubian drainages or other Black Sea basins exhibit mtDNA and allozyme alleles specific to Atlantic brown trout (Bernatchez, Guyomard, and Bonhomme 1992Citation ; Bernatchez and Osinov 1995Citation ). The most likely explanation for this pattern is intergradation of ancient Danubian and Atlantic populations after secondary contact in postglacial times, a pattern that was also corroborated by other studies (e.g., Largiadèr and Scholl 1995Citation ; Weiss et al. 2001Citation ).

Tests of Neutrality
Results from the two-by-two contingency tests do not provide evidence for directional selection. This is because the absence of silent substitutions on PSSs precludes the application of the two-by-two test just to these sites. However, results of the two-by-six matrix test reject the null hypothesis of neutrality, largely because of the PSS and the total absence of silent substitutions. When compared with other sites, this would imply strong directional selection on the PSS. Ford, Thornton, and Park (1999)Citation suggested that positive natural selection for new alleles has played an important role in the evolution of the TF protein in salmonids. The selected sites generally fall on the outside of the molecule, within and near areas that are bound by TF-binding proteins from human pathogenic bacteria, thus supporting the hypothesis that competition for iron could be a source of positive selection (Ford 2001Citation ). Ford (2000)Citation showed that unlike patterns of variation within species, there was no evidence of greater differentiation among chinook salmon (O. tshawytscha) populations at nonsynonymous compared with synonymous sites. Nevertheless, we do find some evidence for directional selection at the intraspecific level in the patterns of TF coding variation within brown trout, and thereby differences in the coalescence patterns among TF lineages may be expected (Takahata 1990Citation ; Stephan and Mitchell 1992Citation ).

Phylogeographic Assemblages of the TF Gene
On the basis of inferences from the TF genealogy and the current spatial distribution of electromorphs, we hypothesize a framework for the evolution of the S. trutta species complex (fig. 8 ). The most ancestral group of populations is characterized by the TF-BCA subclade (present in Black, Caspian, and Aral Sea drainages). This conclusion is further supported by the fossil records. The oldest fossils of the brown trout were found in the Caucasus and date from early Pleistocene, 2 MYA (discussed in Osinov and Bernatchez 1996Citation ). The radiation of the species could have started around this period, or even earlier. Some heterogeneity was observed among this haplogroup, with haplotypes from the Sevan Lake trouts (Caspian drainage) differing by at least nine substitutions from the others. The brown trout from Sevan Lake was initially hypothesized to be a species (S. ischchan) derived from the primitive ancestor of all brown trout populations (Behnke 1986Citation ); however, this view has been contested on the basis of allozyme and mtDNA analyses which suggest that differentiation of this population occurred recently, most probably in late glacial or postglacial times (Bernatchez and Osinov 1995Citation ; Osinov and Bernatchez 1996Citation ). The observed divergence in the TF gene supports a more ancient origin than postglacial times for this population.



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Fig. 8.—(A, B, and C). Scenarios for the origin and migration pathways of the brown trout species complex inferred from the TF gene genealogy. Recombination (REC) and gene conversion (GC) events related to the origin of postrecombinant clades were highlighted on simplified versions of the peeled tree and postrecombinant clades (see figs. 6 and 7 for details) to provide a direct comparison with the inferred phylogeographic scenarios (A, B, and C)

 
The phylogenetic position and accumulated variation of the TF-BCA haplogroup, coupled with its relationship with TF*102, are suggestive of an early radiation of the species and dispersal from western Asia to Europe and an early widespread distribution throughout the Mediterranean region (fig. 8A ). The dispersal could have included movements of individuals through the upper reaches of the Danube system, as has been suggested by the presence of a few DA mtDNA haplotypes in populations from the Adriatic drainages (Bernatchez, Guyomard, and Bonhomme 1992Citation ; Apostolidis et al. 1997Citation ), or the seaway from the Black Sea to the Mediterranean. The latter was suggested by allozyme analyses of Greek and Turkish brown trout populations (Karakousis and Triantaphyllidis 1990Citation ; Togan et al. 1995Citation ; Apostolidis, Karakousis, and Triantaphyllidis 1996Citation ). Population divergence has been achieved later by allopatric fragmentation between the two major drainages.

The TF75 postrecombinant clade was found in the marbled trout (S. trutta marmoratus), which is a phenotypically and ecologically distinct brown trout currently present in Italy, Slovenia, Croatia, and Albania (Adriatic drainages) and typically fixed for TF*75 (Giuffra, Guyomard, and Forneris 1996Citation ; Berrebi et al. 2000Citation ). Based on allozyme data, Giuffra, Guyomard, and Forneris (1996)Citation proposed two alternative hypotheses for the colonization of the Po basin, depending on the order of arrival of the two natural forms that currently occur there (marbled and Mediterranean trout). Further allozyme evidence (Berrebi et al. 2000Citation ) suggests that it was the marbled trout that was the last to arrive in the Adriatic region, and our data corroborate this hypothesis. The high divergence of this postrecombinant clade and the unique TF electromorph exhibited suggest, however, that the marbled trout arrived very early in this region. The presence of mtDNA haplotypes from the MA lineage in three Greek populations from the southern Adriatic-Ionian Sea drainage (Apostolidis et al. 1997Citation ) and the detected gene conversion with the haplogroup BCA suggest that the origin of the postrecombinant clade TF75 could have been to the south of its current occurrence (fig. 8B ). The other subclade from the major terminus T-2, TF95, corresponding to the TF*95 electromorph, exhibits a very distinct range distribution relative to the previous one. At present, TF*95 mainly exists in relic populations of headwater systems. The electromorph is observed, at low frequencies, in the upper Rhône (Mediterranean) and Po (Adriatic) in Switzerland (Largiadèr and Scholl 1995Citation ; Largiadèr, Scholl, and Guyomard 1996Citation ), and at middle to high frequencies in some southwestern Atlantic upper stream populations from Portugal and Spain (Antunes, Alexandrino, and Ferrand 1999Citation ; this study). Antunes, Alexandrino, and Ferrand (1999)Citation reported this puzzling trend and suggested that if common ancestry existed between the TF*95 from these distinct geographic regions, it would indicate the ancestral admixture of distinct geographic groups. Our data show that this electromorph has a rather old ancestry, further corroborated by its occurrence mainly in headwater drainages. The phylogenetic relationship of TF*95 with TF*75, together with their current distribution, suggests that the *95 electromorph went through a long-distance dispersal from the Adriatic region to the southernmost Atlantic regions (fig. 8B ). Because the Adriatic and Mediterranean drainages were already colonized habitats, dispersal was not so successful. By contrast, the Atlantic drainages of the Iberian Peninsula have offered the opportunity to colonize new habitats and continue range expansion to the west.

The major terminus T-3 is characterized by a codon insertion at exon 2. This is a derived brown trout characteristic differing from other salmonid cDNAs (Kvingedal, Aleström, and Rørvik 1993Citation ; Lee et al. 1995Citation , 1998Citation ; Tange et al. 1997Citation ; Ford, Thornton, and Park 1999Citation ; Ford 2000Citation ). The mutation represents the signature of recent successful brown trout dispersal across most of its current distribution range (fig. 8C ). The postrecombinant clade TF78.80 was observed among populations from the Black Sea through the Mediterranean, whereas TF100 was present among the majority of the Atlantic populations. Divergence between these subclades reflects a recombination event and a second major dispersal event from the Mediterranean to the Atlantic drainages. Routes of dispersal do not seem to have been used very frequently, but rather in particular windows of time when climate and geography allowed. TF*78 is fixed for the Italian carpione (S. trutta carpio), an endemic form of Garda Lake (Po basin). This taxon is considered to be of postglacial origin, formed by recent hybridization between marbled trout and Mediterranean trout (Giuffra, Bernatchez, and Guyomard 1994Citation ; Giuffra, Guyomard, and Forneris 1996Citation ). Our results also support the introgressive hybridization origin of this form because we found one Italian carpione exhibiting a sequence related to the marbled trout postrecombinant clade (R3). The similarity in TF sequences between the two individuals from Garda Lake (Adriatic Sea drainage) and one individual from the very remote Kodori River (Black Sea drainage) is puzzling and suggests recent gene flow between these two remote areas. In contrast, we detected some sequence divergence in related haplotypes, corresponding to electromorph TF*80, found in the vicinity of the Adriatic drainage. TF*80 is currently observed in a few Mediterranean French (including Corsica) and Spanish populations (Presa et al. 1994Citation ; Berrebi 1995Citation ; this study). The distribution, both of TF*78 and *80, suggests a low level of penetration in previous established populations of the Adriatic and Mediterranean drainages. In contrast, the widespread distribution of subclade TF100 (corresponding to the electromorph TF*100) throughout the Atlantic drainage suggests a rapid expansion. The Atlantic basin was the one most directly affected by Pleistocene glaciations in terms of habitat loss, with later expansions overriding the range of earlier ones (Hewitt 2000Citation ). The northern part of the Atlantic region was covered with ice during the last glaciation, and thus many populations have existed only since postglacial times (i.e., during the last 10,000–18,000 years); however, the presence of discrete refugia in the southwestern Atlantic regions would have allowed persistence of some populations (e.g. Sanz, García-Marín, and Pla 2000Citation ; Weiss et al. 2000Citation ; Antunes et al. 2001Citation ; Bouza et al. 2001Citation ). This would explain part of the complexity found in the southernmost Atlantic populations, with the maintenance of some relic populations carrying the subclade TF95, and some structure within the subclade TF100.

Additional complexity in the brown trout evolutionary history was then added by the intergradation of differentiated subclades and postrecombination clades, as detected by some of the recombination-gene conversion events. Previous studies based on allozymes and mtDNA data identified introgressive hybridization between different lineages in glacial or postglacial times (e.g., Bernatchez, Guyomard, and Bonhomme 1992Citation ; Largiadèr and Scholl 1995Citation ; Osinov and Bernatchez 1996Citation ); however, secondary intergradation (excluding that caused by stocking) seems to have been relatively limited (e.g., Bernatchez and Osinov 1995Citation ; Giuffra, Guyomard, and Forneris 1996Citation ). Biological factors, in addition to physical isolation, have probably limited dispersal and introgressive hybridization as can be inferred by the existence of five mtDNA lineages that evolved in geographic isolation during the Pleistocene and have remained largely allopatric since then (Bernatchez 2001Citation ; fig. 1 ).

Levels of Congruence with mtDNA Variation
The TF genealogy was consistent with some of the phylogenetic lineages recognized with the mtDNA. The brown trout from the BCA region (comprising populations geographically corresponding to the DA mtDNA lineage) seems to be mainly characterized by the TF-BCA haplogroup. The marbled trout (MA mtDNA lineage) is also characterized by just one postrecombinant clade (TF75). However, brown trout from other geographic areas exhibited TF haplotypes from different subclades. This is the case of brown trout from the Mediterranean and Adriatic basins (ME and AD mtDNA lineage, respectively) exhibiting the TF102 and TF78.80 subclades. Populations from the Atlantic basin (AT mtDNA lineage) exhibited the TF95 and TF100 subclades. The phylogeographic scenario inferred from the TF genealogy seems to predate that of the mtDNA. Whereas the mtDNA alleles have had sufficient time to sort to reciprocal monophyly, the alleles at the TF locus have not. Because mtDNA is haploid and maternally transmitted, it is expected to have a fourfold lower Ne than autosomal nuclear loci (Birky, Fuerst, and Maruyama 1989Citation ). Under neutral models, the genetic drift governs the process of lineage sorting, and smaller effective population sizes (Ne) lead, on an average, to higher rates of stochastic lineage extinction and fixation (Hoelzer 1997Citation ). This could also be a reason for the low mtDNA haplotype diversity found in the ME and MA lineages (three and four distinct haplotypes, respectively, in 104 and 205 individuals analyzed; Bernatchez 2001Citation ). In contrast, our results, focusing on a small number of individuals, show a considerable number of distinct TF haplotypes corresponding to the major electromorphs that characterize these population groups.

The phylogenetic relationships of the TF gene show that haplotypes from the BCA region were those, among brown trout, exhibiting the highest similarity with the outgroup (fig. 7 ). Some of this homology results from RGCs, which are character states that arise rarely and are not subjected to extensive convergent or parallel evolution, contributing to a low degree of homoplasy (Rokas and Holland 2000Citation ). At the mtDNA level, the ancestral divergence of the AT lineage from all others was suggested when considering the Atlantic salmon as the outgroup (Giuffra, Bernatchez, and Guyomard 1994Citation ; further discussed in Bernatchez 2001Citation ) (fig. 1 ). The distinctness of the AT group was determined by the identity at four nucleotide positions with S. salar that differed in all other S. trutta genotypes, based on pooled sequences (1.25 kb) of both the control region and coding genes (cytochrome b and ATPase subunit VI). Assuming that the AT clade split from the ME or a common ancestor after the Messinian crisis (Machordom et al. 2000Citation ), Antunes et al. (2001)Citation used a control region fragment of 464 bp and observed two statistically parsimonious solutions for the connecting branch of the two lineages. This suggests that because of the higher mutation level of mtDNA, even more homoplasy could be expected when comparisons are made at the generic level. According to our data, the TF100 subclade corresponding to the TF*100 electromorph that currently characterizes the majority of Atlantic populations is the most divergent from the outgroup.


    Conclusions
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
In summary, the TF gene genealogy presented here clearly provides new phylogeographic insights over previous allozyme and mtDNA studies and shows the potential utility of nuclear genes for intraspecific evolutionary inferences. Furthermore, intragenic recombination events provide evidence of past secondary intergradation and indicate the improved utility of nuclear markers as tools for recovering complex evolutionary histories. Incomplete congruence between nuclear and mitochondrial phylogeographic patterns is best explained by different modes of evolution and transmission. Moreover, a variety of reasons involving demography, history, selection, and independent DNA sequences within the same organismal pedigree may result in quite different phylogeographic patterns (Hare and Avise 1998Citation ). Thus, accurate estimation of the evolutionary history of an organism should use different loci, avoiding inferences confounded by the inherent differences between maternally and biparentally inherited genes.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
This work was financed in part by the Fundação para a Ciência e a Tecnologia (FCT) Projects (Praxis XXI/P/BIA/10245/1998 and PRAXIS XXI/P/BIA/11174/1998). A.A. was supported by a Ph.D. grant (Praxis XXI/BD/11003/97) and by a postdoctoral grant (SFRH/BPD/5700/2001), both from FCT. We thank P. Berrebi, J. L. García-Marín, E. García-Vázquez, P. Martínez, A. Osinov, and S. Weiss for providing samples. Several discussions with E. Eizirik, N. Ferrand, W. E. Johnson, J. Martenson, and W. J. Murphy were important for this work. Comments made by J. W. Arntzen, M. Branco, the Associate Editor K. Crandall, and two anonymous referees improved a previous version of this manuscript.


    Footnotes
 
Keith Crandall, Reviewing Editor

Keywords: brown trout gene conversion nuclear genealogy recombination Salmo trutta salmonids transferrin Back

Address for correspondence and reprints: Agostinho Antunes, Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland 21702-1201. aantunes{at}ncifcrf.gov , aantunes{at}hotmail.com Back


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Accepted for publication March 20, 2002.