Present addresses: A. P. Michel, Department of Entomology, Ohio Agricultural Research and Development Center, Ohio State University, 210 Thorne Hall, 1680 Madison Ave., Wooster, OH 44691, USA. D. Schwarz, Department of Entomology, University of Illinois at Urbana-Champaign, Morrill Hall, 505 S. Goodwin Ave., Urbana, IL 61801, USA. S. Velez, Museum of Comparative Zoology, Harvard University, 26 Oxford St, Cambridge, MA 02138, USA.
Radiation and divergence in the Rhagoletis Pomonella species complex: inferences from DNA sequence data
Article first published online: 25 FEB 2008
© 2008 The Authors
Journal of Evolutionary Biology
Volume 21, Issue 3, pages 900–913, May 2008
How to Cite
XIE, X., MICHEL, A. P., SCHWARZ, D., RULL, J., VELEZ, S., FORBES, A. A., ALUJA, M. and FEDER, J. L. (2008), Radiation and divergence in the Rhagoletis Pomonella species complex: inferences from DNA sequence data. Journal of Evolutionary Biology, 21: 900–913. doi: 10.1111/j.1420-9101.2008.01507.x
- Issue published online: 25 FEB 2008
- Article first published online: 25 FEB 2008
- Received 1 November 2007; revised 19 December 2007; accepted 19 December 2007
- differential introgression;
- genetic divergence;
- host races;
- sibling species;
- speciation mode plurality;
- sympatric speciation
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- Supporting Information
Here, we investigate the evolutionary history and pattern of genetic divergence in the Rhagoletis pomonella (Diptera: Tephritidae) sibling species complex, a model for sympatric speciation via host plant shifting, using 11 anonymous nuclear genes and mtDNA. We report that DNA sequence results largely coincide with those of previous allozyme studies. Rhagoletis cornivora was basal in the complex, distinguished by fixed substitutions at all loci. Gene trees did not provide reciprocally monophyletic relationships among US populations of R. pomonella, R. mendax, R. zephyria and the undescribed flowering dogwood fly. However, private alleles were found for these taxa for certain loci. We discuss the implications of the results with respect to identifiable genetic signposts (stages) of speciation, the mosaic nature of genomic differentiation distinguishing formative species and a concept of speciation mode plurality involving a biogeographic contribution to sympatric speciation in the R. pomonella complex.
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- Supporting Information
Understanding what a species is and the processes responsible for their formation are two central issues in evolutionary biology. The standard answer to the first question has been the biological species concept (BSC), which defines species on the basis of intrinsic barriers to gene flow (Mayr, 1963). Geographic isolation (allopatry) has been argued to be the common denominator with respect to answering the second question of speciation mode (Mayr, 1963).
In recent years, a number of alternative, genetically based species concepts have been proposed to the BSC. These concepts include defining species based on the existence of genotypic clusters in sympatry (Mallet, 1995; Feder, 1998), on the presence of diagnostic autapomorphies distinguishing populations (Cracraft, 1989) and on reciprocal monophyly between evolutionary lineages (de Queiroz, 1998). Although debate continues on the merits, practicality and relationship of these genetic concepts relative to the BSC (Coyne & Orr, 2004), the discussion has served to highlight that the genomes of diverging populations, particularly taxa undergoing divergence-with-gene-flow speciation, can be mosaic. Regions containing loci under selection contributing to gene flow barriers (islands of speciation; Turner et al., 2005) can display greater differentiation than regions not involved in reproductive isolation. A single value of reproductive isolation between taxa may therefore not apply uniformly across the genome (Mallet, 1995; Feder, 1998; Wu, 2001), as whole genomes need not be isolated (Mayr, 2001) or monophyletic (Hudson & Coyne, 2002) to have good species.
The issue of speciation process has also been contentious. Recently, however, a growing consensus has emerged that allopatry is not the exclusive mode of speciation, even for animals; divergence-with-gene-flow speciation can and does occur, although under more restrictive circumstances than speciation in allopatry (Coyne & Orr, 2004). An important accompanying development in this discussion has been an increasing awareness of the possibility of speciation mode ‘plurality’; that categorizing speciation as strictly allopatric vs. nonallopatric may not adequately describe the geographic context of divergence for many taxa. If, for example, some genetic changes leading to reproductive isolation occur in allopatry and others in sympatry, then it would seem appropriate for the geographic mode of speciation to be mixed (Mallet, 2005). In this regard, Coyne & Orr (2004) have proposed several new terms describing situations of mixed geographic mode.
The Rhagoletis pomonella (Diptera: Tephritidae) complex provides an opportunity to investigate questions concerning speciation pattern and process. The complex contains a number of host races (e.g. apple and hawthorn-infesting populations of R. pomonella) and sibling species (e.g. R. mendax, R. zephyria, R. cornivora and the undescribed flowering dogwood fly) at varying stages of divergence. The close morphological similarity, distinct host plant affiliations and broadly overlapping geographic ranges of R. pomonella complex flies led Bush (1966, 1969) to propose that its members all speciated sympatrically via host plant shifting in North America. The central premise of the sympatric hypothesis is that differential adaptation to alternative host plants generates ecological barriers to gene flow that initiate speciation. For R. pomonella, each fly in the complex infests a unique, nonoverlapping set of host plants (see supplementary Table S1) which tend to fruit at different times of the field season (Smith, 1988; Feder et al., 1993; Berlocher, 2000; Dambroski & Feder, 2007). Because Rhagoletis is univoltine, differences in the depth of the over-wintering diapause differentially adapts flies to variation in host fruiting phenology, generating allochronic mating isolation between R. pomonella taxa (Smith, 1988; Feder et al., 1993, 1994; Dambroski & Feder, 2007). Rhagoletis flies also mate almost exclusively on or near the fruit of their respective host plants (Prokopy et al. 1971, 1972). Traits related to host plant discrimination, including differences in fruit volatile preference, therefore also directly affect mate choice and represent another important ecological barrier to gene flow among R. pomonella flies (Feder et al., 1994; Linn et al., 2003, 2004).
Here, we investigate patterns of DNA sequence divergence for 11 anonymous nuclear genes and mtDNA in the R. pomonella complex. The objectives of the study are threefold. Our first goal is to test whether any ‘genetic signposts’ of divergence exist for R. pomonella flies. By genetic signpost, we are referring to identifiable patterns of differentiation, such as degree of genotypic clustering or monophyly, in gene trees indicative of stages (the extent of ecological and intrinsic isolation) in the speciation continuum, rather than specific conditions that have to be met to satisfy a particular species concept. With the exception of R. cornivora, previous allozyme and mtDNA work has failed to uncover diagnostically fixed allele differences distinguishing any of the other taxa (McPheron et al., 1988; Berlocher et al., 1993; Feder, 1998; Feder et al., 1999; Berlocher, 2000). Some taxa, such as R. mendax and R. zephyria, do possess high-frequency allozyme variants that are rare in other populations (Berlocher et al., 1993; Feder, 1998; Feder et al., 1999; Berlocher, 2000), but they are not diagnostic. This has lead to speculation that certain R. pomonella taxa may represent ‘quantitative genetic’ species; species that display marked phenotypic differences in host-related ecological adaptations causing substantial reproductive isolation because of the cumulative effects of significant allele frequency, but not fixed differences, across contributing loci (Berlocher & Feder, 2002). If more sensitive sequence analysis reveals the same pattern, then we must begin to ask why fixed differences are absent; are most of the sibling species too young for the lineage sorting of neutral variation to be complete or is persistent, low-level gene flow among taxa sufficient to prevent alleles from becoming fixed?
Our second goal was to determine whether gene trees for the nuclear loci are consistent with a hypothesis of speciation mode plurality for R. pomonella. Earlier work has suggested that introgression from Mexico helped facilitate the sympatric radiation of the R. pomonella complex in the USA (Feder et al., 2003a, 2005; Xie et al., 2007). Sometime during a period of past geographic separation ∼1.57 Ma, inversions appeared to have arisen and fixed in an isolated hawthorn fly population located in the Eje Volcanico Trans Mexicano (EVTM; Fig. 1) for three different genomic regions on chromosomes 1, 2 and 3 (haploid n = 6 for R. pomonella; Bush, 1966). Following secondary contact, gene flow from the EVTM into the USA probably through the conduit of the Sierra Madre Oriental Mountains (SMO in Fig. 1) created adaptive inversion clines for diapause life-history traits in the USA (Xie et al., 2007). This latitudinal diapause variation in conjunction with additional changes in host discrimination aided the US hawthorn fly population in sympatrically shifting and adapting to a variety of new plants with differing fruiting times and other physical and chemical characteristics, generating R. mendax, R. zephyria and the flowering dogwood fly in the process. In historical time (< 150 years), R. pomonella also shifted from hawthorn to introduced, domesticated apple (Malus pumila) resulting in the formation of the apple fly race in the north-eastern and mid-western USA (Bush, 1966). Therefore, a portion of the diapause variation contributing to the radiation of the R. pomonella complex in the USA originated at an earlier time and in a different location than the sympatric host shifts triggering speciation. In this study, we increase the sampling of nuclear loci and taxa to confirm the chronology of the isolation of Mexican EVTM flies relative to the radiation of the R. pomonella complex in the USA.
Our third goal is to determine whether patterns of genetic divergence vary across the R. pomonella genome. Previous work implied that the inverted regions of chromosomes 1–3 display greater levels of divergence between Mexican and US hawthorn flies than loci mapping elsewhere on chromosomes 4 and 5 (Feder et al., 2005; Xie et al., 2007). From these data, we inferred that after the initial period of introgression from Mexico, the rearranged regions of chromosomes 1–3 may have evolved to become more impervious to gene flow than loci residing on other chromosomes. Through increased sampling of sibling taxa, we investigate whether a similar pattern is evident for the other US members of the R. pomonella complex as well.
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- Supporting Information
Flies and collection sites
Rhagoletis pomonella complex flies were genetically analysed from 14 different sites/ populations, 10 from the USA and four from Mexico (Fig. 1; see supplementary Table S1 for complete details of the sites, including host species and collecting dates). Two of the four Mexican hawthorn sites [Coajomulco, Morelos (Evtm CJ) and Tancitaro, Michoacan (Evtm MC)] reside in the Eje Volcanico Trans Mexicano plateau (green outlined area in Fig. 1). The other two Mexican hawthorn sites [San Joaquin, Queretaro (Smo SJ) and Piletas, Veracruz (Smo PL)] reside in the Sierra Madre Oriental Mountains, and are located along the hypothesized conduit for introgression from Mexico into the USA (orange area in Fig. 1). We also sequenced Rhagoletis electromorpha from Dowagiac, MI, USA. to serve as an outgroup (R. electromorpha belongs to the tabellaria species complex, the sister group to R. pomonella; Bush, 1966). Flies were sampled as larvae in infested fruit at all sites and either immediately dissected from the fruit and frozen for later genetic analysis or reared to adulthood in the laboratory.
Sequence data were generated for 11 anonymous nuclear loci and a 626-bp fragment of the mitochondrial cytochrome oxidase II gene (see Table 1 and supplementary information for details; accession numbers in GenBank for DNA sequences are AY152477–AY152526, AY930466–AY931013, DQ812553–DQ812885 and EU108879–EU109174). Six of the 11 loci (P181, P3072, P2956, P667, P7 and P22) map to chromosomes 1–3 and are subsumed by inversions (Roethele et al., 2001; Feder et al., 2003b). Four of the remaining five loci analysed in the study (P661, P2963, P1700 and P309) map to chromosomes 4 and 5 (Table 1). The exact map position of P3060 is not known, but it is not located on chromosomes 1–3. The 11 loci analysed in the current study are therefore broadly representative of R. pomonella genome with respect to map position (all five major autosomes were sequenced for at least two genes each), location within rearranged vs. collinear regions and the potential for host-related selection and introgression. Consequently, if fixed genetic differences do exist among R. pomonella complex flies, there are reasonable expectations that they should be revealed by the current survey.
|P181||1||288||1.000||TrN + I||2|
|P2956||2||510||0.244||TrN + I||5|
|P667||2||583||0.866||TVM + G||5|
|P7||3||736||0.576||TrN + I||2|
|P2963||4||377||0.756||GTR + G||3|
|P661||4||436||0.088||TVM + G||6|
|P1700||5||484||0.997||HKY + G||6|
|P309||5||482||1.000||K81uf + G||3|
|mtDNA||–||626||0.990||TrN + I||0|
DNA cloning and sequencing
Genomic DNA were isolated from individual flies and PCR amplified for 35 cycles (94 °C, 30 s; 52 °C, 1 min; 72 °C, 1.5 min) using locus-specific primers for the 11 nuclear and mtDNA fragments as described in Roethele et al. (2001). Products were TA cloned into pCR II vectors (Invitrogen, Calsbad, CA, USA). PCR amplification products were initially cloned separately for a minimum of two flies from each study site, with an attempt made to sequence four to six clones per locus per fly in both the 5′- and 3′-directions on an ABI 3700 sequencer using the ABI Prism® BigDyeTM Terminator v3.0 system (Applied Biosystems, Foster City, CA, USA). To try and increase sample sizes for certain sites, we also separately amplified genomic DNA for four to eight flies from the site, and TA cloned the pooled amplification products for sequencing. To avoid analysis of identical alleles from the same individual, sequences generated from the pooled library were not included unless they differed from each other. We were unable to generate reliable sequence data for the Brazos Bend, Texas (Pom TX) and Geneva, New York (Pom NY) hawthorn-infesting R. pomonella population for the locus P1700. In addition, we only sequenced R. mendax and R. zephyria flies from the Pennsylvania (PA) sites for P1700 and mtDNA.
Gene tree construction and analysis
Parsimony and maximum-likelihood gene trees were constructed using PAUP*b10 (Swofford, 2002). For the parsimony analysis, gaps were treated as a fifth base pair, with indels of identical length and sequence position recoded to count as single mutational steps. Rhagoletis electromorpha was used as an out-group taxon to root trees. Parsimony and maximum-likelihood trees were very similar and so we report the results for only the parsimony trees here. Intragenic recombination was statistically tested using the methods of Hudson & Kaplan (1985). The molecular clock was tested for each locus for R. pomonella and R. electromorpha sequences by comparing log-likelihood scores enforcing vs. relaxing the clock hypothesis for the best supported DNA substitution model identified using MODELTEST (Posada & Crandall, 1998).
Neighbour-joining trees (Saitou & Nei, 1987) summarizing the overall genetic relatedness of populations were constructed using PHYLIP v 3.66 (Felsenstein, 1989). Trees were constructed separately for the six loci mapping to chromosomes 1–3 plus P1700 and for the remaining four loci residing elsewhere in the genome. To construct the neighbour-joining trees, mean pairwise uncorrected genetic distances were first computed separately for each locus between each pair of populations (except the PA sites), as well as between these populations and the outgroup R. electromorpha using Mega v3.1 (Kumar et al., 2004). The pairwise distance between two populations for a locus was then divided by the average distance of all R. pomonella populations to R. electromorpha to standardize for sequence length and substitution rate differences among loci. For the two SMO populations, two diverged haplotypes could generally be identified at loci, one more closely related to Mexican EVTM alleles and the other to SN alleles from the USA (see Figs 2–4 and supplementary Figs S1–S4). We considered these haplotype classes as separate SMO populations in the calculations of genetic distance. Similarly, for the chromosome 1–3 loci and P1700, taxa possessing North (N) and South–North (SN) haplotypes at a locus were considered different populations in genetic distance calculations. Standardized distances were then averaged across P181, P3072, P2956, P667, P7, P22 and P1700 and across P661, P2963, P309 and P3060 to give the overall pairwise distances used for tree construction. Because of the variable presence of N haplotypes in taxa other than R. pomonella, we only included the N haplotype population from NY as a general representative of the N clade in the network.
Unfortunately, other analytical approaches, such as the isolation model with migration (IM or IMa; Nielsen & Wakeley, 2001; Hey & Nielsen, 2007), are not readily applicable to the R. pomonella data set because of violations of underlying assumptions of the model, including that there be no unsampled populations exchanging genes with the sampled populations or their ancestor, no directional or balancing selection acting on sites (selective neutrality), no recombination within loci and free recombination between loci.
Analysis of population differentiation and structure
A hierarchical analysis of molecular variance (amova) was performed using Arlequin 2.0 (Schneider et al., 2000) to test for genetic structuring among populations. For the amovas, populations were divided into six groups: (1) EVTM R. pomonella sites (MC and CJ); (2) SMO R. pomonella sites (SJ and PL); (3) US R. pomonella sites (Pom MI, NY and TX); (4) flowering dogwood fly sites (Dog IN and GA); (5) R. mendax (Mend MI site); and (6) R. zephyria (Zeph WA site). Separate amovas were performed for the six chromosome 1–3 loci and P1700 with N haplotypes both excluded (−N) and included (+N). Neither R. cornivora nor R. electromorpha were included in the amovas, as gene trees showed these taxa to be basal and their inclusion would have inflated the per cent variation explained by among-group differences for the other ‘in-group’ taxa in the analysis. Tamura & Nei (1993) genetic distances between in-group taxa and R. electromorpha for loci were calculated using Mega v3.1 (Kumar et al., 2004).
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- Supporting Information
None of the 11 sequenced nuclear genes or mtDNA deviated significantly from a molecular clock (Table 1). All nuclear loci except P3060 displayed evidence for possible recombination, as implied by the method of Hudson and Kaplan (Table 1). Inferred recombination was generally limited, however, to alleles within the same haplotype class for loci on chromosomes 1–3 or same geographic population/taxa. There was no evidence for recombination among mtDNA sequences (Table 1). Confirmation of clock-like evolution and intra-haplotype-limited recombination allowed for inferences to be drawn concerning the three goals of the study based on comparisons of branching topologies and node depths among gene trees. We organize the presentation of these results below into three subsections highlighting the similarities and differences in patterns of genetic differentiation for: (1) the six loci on chromosomes 1–3 (genes residing in inverted regions of the genome); (2) the five loci mapping to chromosomes 4 and 5 (genes in putative co-linear regions of the genome); and (3) mtDNA.
Genes on chromosomes 1–3
Several features characterized the gene trees for the six loci mapping to chromosomes 1–3 (Fig. 2a–c and supplementary Figs S1–S3). First, R. cornivora (designated by R. corn. in the figures) was clearly basal to the other R. pomonella populations, displaying fixed, autapomorphic substitutions for P181, P3072, P2956, P667, P7 and P22.
Second, with the exception of P7, gene trees for chromosome 1–3 loci resolved US and Mexican R. pomonella, R. zephyria, R. mendax and the flowering dogwood fly as a distinct monophyletic ‘in-group’ clade from R. cornivora (Fig. 2a–c and supplementary Figs S1–S3).
Third, within the ‘in-group’ taxa of US and Mexican R. pomonella populations, R. zephyria, R. mendax and the flowering dogwood fly, two distinct clades/classes of haplotypes could be identified for each locus. One clade consisted of North haplotypes (designated by the blue-coloured letter ‘N’ in Fig. 2a–c and supplementary Figs S1–S3) found in northern populations of R. pomonella (NY and MI), and to varying degrees, depending on the locus, in R. mendax, R. zephyria and the flowering dogwood fly as well. The other clade was comprised of a trinity of haplotypes present: (1) in northern and southern populations of US R. pomonella, R. mendax, R. zephyria and the flowering dogwood fly (designated by the red-coloured letters ‘SN’ in the figures); (2) in the Sierra Madre Oriental population of Mexican hawthorn flies (designated by the orange-coloured letters ‘SMO’); and (3) in the Eje Volcanico Trans Mexicano region (designated by the green-coloured letters ‘EVTM’).
Fourth, the depths of the nodes uniting the N clade and the triad of SN/SMO/EVTM haplotypes relative to the outgroup R. electromorpha were similar among loci and concurred with a deep division seen for mtDNA between EVTM flies in Mexico and the remainder of the in-group taxa (compare yellow-coloured nodes in Fig. 2a–c and supplementary Figs S1–S3 among loci). The congruence of these nodes among gene trees is hypothesized to reflect the initial isolation of the Mexican EVTM population that predated the secondary contact and introgression of nuclear SN haplotypes into the USA.
Fifth, aside from P7, the Mexican population of hawthorn-infesting R. pomonella in the EVTM designated by the green-coloured haplotypes was monophyletic and displayed fixed, autapomorphic differences from the other taxa for the five other nuclear loci on chromosomes 1–3 (Fig. 2a–c and supplementary Figs S1–S3).
Sixth, the hawthorn-infesting populations of R. pomonella in the SMO generally contained a mixture of alleles that showed affinity to both the EVTM and the USA. For P3072 and P667, SMO flies possessed EVTM haplotypes, forming a monophyletic clade (Fig. 2a and supplementary Fig. S2). For P22 and P181, SMO flies had alleles embedded within the SN class of haplotypes present in US R. pomonella, R. mendax, R. zephyria and flowering dogwood fly populations (Fig. 2b and supplementary Fig. S1). The locus 2956 had haplotypes related to both EVTM and the SN clades of alleles (Fig. 2b). Finally, P7 (supplementary Fig. S1) contrasted with the other chromosome 1–3 genes in that SMO alleles formed a distinct clade genealogically unrelated to either EVTM or SN haplotypes.
Seventh, no fixed diagnostic substitution distinguished R. pomonella, R. mendax, R. zephyria and the flowering dogwood fly in the USA from each other for P3072, P181, P2956, P667 or P7, although an autapomorphy possibly exists for R. zephyria at P22 (Fig. 2a–c and supplementary Figs S1–S4). However, private alleles were observed at several genes for R. mendax and R. zephyria, and much less so for the flowering dogwood fly.
The neighbour-joining network based on overall relative genetic distance measures among populations for chromosome 1–3 loci and P1700 (Fig. 3a) highlighted the general patterns discerned from the individual gene trees, including: (1) the basal position for R. cornivora in the pomonella complex; (2) the presence of a deep northern clade of alleles (designated by Pom-NY) within the in-group taxa in the USA; (3) the existence of a distinct, monophyletic clade of Mexican hawthorn fly haplotypes in the EVTM; and (4) the composite nature of the Mexican hawthorn fly population in the SMO, containing genetic elements with affinity to both EVTM and US haplotypes. The network also inferred that R. pomonella flies in the USA are overall genetically most similar to flowering dogwood flies, followed by R. zephyria, and then R. mendax. In addition, the class of alleles within the SMO showing affinity to SN haplotypes in the USA is most closely related to R. pomonella, and, in particular, the TX population.
Genes mapping outside chromosomes 1–3
The five loci mapping outside chromosomes 1–3 were similar to the six chromosome 1–3 loci in several respects. Gene trees for P661, P2963, P1700, P309 and P3060 clearly positioned R. cornivora basal to the other members of the R. pomonella complex (Fig. 4a–d and supplementary Fig. S4). The in-group taxa comprised of US and Mexican R. pomonella, R. zephyria, R. mendax and the flowering dogwood fly were also monophyletic for P661, P2963, P1700, P309 and P3060, sharing several synapomorphic substitutions in common. In addition, P661, P2963, P1700, P309 and P3060 showed substantial genetic differentiation among R. pomonella populations, as indicated by significant FCT values in a hierarchical amova, similar to the six chromosome 1–3 loci (Table 2). Indeed, P1700 displayed two potentially fixed autapomorphic substitutions diagnostically distinguishing R. zephyria from all other R. pomonella complex flies (Fig. 4c). The flowering dogwood fly also possessed a clade of private haplotypes for P1700, as did R. mendax for the loci P309 and P1700 (Fig. 4a) and R. zephyria for P661 (supplementary Fig. S4).
|Locus||Chr.||N||Percent variation explained||Fixation indices|
|Among pops.||Among sites in pops.||Within sites||FCT (pop./total)||FSC (site/pop.)||FST (site/total)|
There were several pronounced differences, however, between loci residing on chromosomes 1–3 and most genes mapping elsewhere in the genome. First, except for P1700, no locus mapping outside of chromosomes 1–3 possessed a highly diverged N haplotype class of alleles (Fig. 4b–d and supplementary Fig. S4). Second, no monophyletic clade of Mexican EVTM haplotypes was present for P309, P3060 or P661 (Fig. 4a,b and supplementary Fig. S4). Third, Mexican SMO alleles for P309, P3060 and P661 were not distinct and were embedded in the gene tree with SN alleles from the USA; these three loci provided no evidence for the SMO having genetic affinity to the EVTM. Finally, although a significant proportion of the genetic variation present for P309, P3060, P2963 and P661 was partitioned among taxa, the relative degree of genetic divergence separating the alleles displaying variation was less for these loci than for the six loci residing on chromosomes 1–3 (compare the depths of the black-coloured circles signifying the nodes uniting EVTM and SN between gene tree in Fig. 4 and supplementary Fig. S4 vs. Fig. 2 and supplementary Figs S1–S3, as well as between the neighbour-joining networks in Fig. 3a,b; note: P1700 was included with the six chromosome 1–3 loci for network construction because of the observation of distinct N and SN haplotypes at the locus). The shallower divergence among alleles for nonchromosomes 1–3 loci was not because of a slower rate of evolution for these genes (mean Tamura–Nei distance for in-group R. pomonella taxa to R. electromorpha for loci not on chromosomes 1–3 = 0.053 ± 0.0152, n = 4, range 0.032–0.066; mean for chromosome 1–3 loci = 0.046 ± 0.0222, n = 6, range 0.020–0.085; distance for P1700 = 0.056 ± 0.0104). Moreover, the gene trees in Figs 2 and 4 are all scaled relative to R. electromorpha and the networks in Fig. 3 are based on relative, not raw, genetic distances to R. electromorpha. Rather, the difference reflects the greater genetic similarity of P661, P2963, P309 and P3060 alleles among the in-group R. pomonella taxa than P181, P3072, P2956, P667, P7, P22 and P1700 haplotypes. As a consequence, the neighbour-joining network for P661, P2963, P309 and P3060 (Fig. 3b) was less well resolved than that for the chromosome 1–3 loci and P1700 (Fig. 3a), with US R. pomonella and flowering dogwood fly populations appearing paraphyletic and R. zephyria being embedded within the Mexican SMO populations.
MtDNA displayed both similarities and differences from the patterns of genetic variation observed for the nuclear loci. Rhagoletis cornivora was again basal in the R. pomonella complex. Like P661, P2963, P309 and P3060, mtDNA did not display a highly diverged N haplotype confined to northern US fly populations (Fig. 2d). However, the EVTM and the in-group R. pomonella taxa showed a deep genetic divergence for mtDNA that was congruent with the relative node depths between N and SN/SMO/EVTM clades of haplotypes for chromosomes 1–3 loci (Fig. 2). However, there was no signature for subsequent introgression in the mtDNA between the EVTM and US or SMO fly populations. Moreover, unlike the chromosome 1–3 loci, but like P661, P309 and P3600, mtDNA, haplotypes for SMO flies were not a mixture of EVTM and US types. Instead, they were all embedded within and very similar to SN haplotypes found in the USA (Fig. 2d). There was no diagnostic mtDNA autapomorphy distinguishing R. pomonella, R. mendax, R. zephyria, the flowering dogwood fly or SMO flies for COII (Fig. 2d). However, R. mendax and R. zephyria appeared to possess potential private substitutions and, on this basis, were the most genetically differentiated of the in-group taxa for mtDNA.
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Genetic signposts for speciation
The first goal of the current sequence study was to determine whether genetically distinguishable stages of population divergence could be identified in the R. pomonella complex. Our results suggest that on a coarse, qualitative scale such stages may be recognized with respect to: (1) R. cornivora (fixed, reciprocal monophyly for all loci); (2) EVTM hawthorn flies (fixed substitutions and monophyly for several, but not all loci); (3) SMO hawthorn flies (fixed differences for a couple of loci and some private alleles for a handful of genes); (4) the in-group sibling taxa in the USA (possible autapomorphies and private alleles at a few loci); and (5) the apple and hawthorn host races (no fixed substitution or private allele difference). The sequence data did not reveal any finer genetic resolution than previous allozymes studies, however (Feder & Bush, 1989; Berlocher et al., 1993; Feder, 1998; Feder et al., 1999; Berlocher, 2000). Moreover, neither nuclear or mtDNA loci clearly resolved the phylogenetic relationships (i.e. a bifurcating gene tree) among the in-group populations of R. pomonella, R. mendax, R. zephyria and flowering dogwood fly in the USA. Reciprocal monophyly was not observed for any locus among these four in-group taxa and it is conceivable that with increased sampling the potential private and autapomorphic variants seen for R. mendax and R. zephyria for a couple of genes in the current study will prove to be shared among taxa, albeit at low frequencies, as is the case for allozymes. Such an outcome is suggested by RFLP data for P1700 for R. mendax and R. zephyria (Schwarz et al., 2005). The sequence analysis for R. pomonella flies therefore did not codify a clear demarcation of taxonomic status for US populations based on phylogenetic or lineage concepts of species, although the neighbour-joining network based on overall relative genetic distances measures for chromosome 1–3 loci and P1700 did imply that R. pomonella and the flowering dogwood fly are sister taxa, followed by R. zephyria and R. mendax (Fig. 3a).
There are two interpretations for the general lack of phylogenetic resolution of the DNA data for the USA in-group taxa. The first explanation is that diagnostic differences exist, but that the sequencing of 11 nuclear loci and mtDNA was not an extensive enough survey of the genome to detect these differences. The current data for the in-group taxa therefore mainly reflect the consequences of incomplete lineage sorting among the relatively recently diverged in-group fly populations in the USA. The second interpretation is that the over 30 allozymes and 11 nuclear loci analysed to date actually do represent an accurate snapshot of the state of genetic differentiation among the in-group R. pomonella taxa. If true, then these taxa are probably best characterized as a continuum of quantitative genetic sibling species and host races distinguishable as sympatric genotypic clusters (Mallet, 1995) on the basis of near private mutations and/or allele frequency differences (Feder, 1998). Members of the group would therefore reflect a dynamic equilibrium between low-level gene flow and the strength of differential host selection, forming a complex analogous to the syngameons of plants. The recent findings of Schwarz et al. (2005) of an historical hybridization event between R. mendax and R. zephyria potentially giving rise to a new race/species of fly in the eastern USA infesting introduced honeysuckle from the Lonicera tatarica complex are consistent with this dynamic equilibrium view for the R. pomonella group.
Although the current data failed to reveal reciprocal monophyly among the R. pomonella in-group taxa in the USA, there were several a priori reasons to believe that the 11 anonymous nuclear loci sequenced in the study should have been sufficient to uncover fixed, diagnostic differences (autapomorphies) provided they exist. First, the in-group taxa R. pomonella, R. mendax, R. zephyria and the flowering dogwood fly possess differential host-associated adaptations related to diapause and host discrimination that serve as significant ecological barriers to gene flow (Smith, 1986; Bierbaum & Bush, 1990; Feder et al., 1993, 1994; Linn et al., 2003; Schwarz et al., 2007). Second, R. pomonella × R. mendax and R. mendax × R. zephyria display a degree of nonhost-related prezygotic isolation. In particular, male R. mendax have difficulty mating with female R. pomonella because of a difference in adult body size between the species (Smith, 1986; J. L. Feder, pers. obs.) and even the similar-sized R. mendax and R. zephyria show significant sexual isolation (Schwarz & McPheron, 2007). Third, R. pomonella and R. mendax also exhibit post-zygotic isolation (over 50%) that could be because of intrinsic genomic incompatibility and/or host fruit-related larval survivorship differences (Smith, 1986; J. L. Feder, pers. obs.). No prezygotic isolation appears to exist between R. pomonella and the flowering dogwood fly. However, there is subtle evidence for possible low-level post-zygotic isolation. Although there is no reduction in fecundity in apple race × dogwood fly matings, backcrosses of F1 hybrids to parental taxa suggest that egg hatch may be reduced by 10% for second generation flies of mixed apple/dogwood ancestry (Smith, 1986). Fourth, although there is also no evidence for reduced fecundity or fertility in hybrid crosses between the apple and hawthorn host races of R. pomonella (Reissig & Smith, 1978; Smith, 1986), F1 apple × hawthorn flies fail to orient to host fruit volatiles in flight tunnel assays (Linn et al., 2004; Dambroski et al., 2005) indicative of an impaired olfactory system that could have significant fitness consequences in the field. Fifth, the 11 nuclear loci we sequenced in the study representatively cover five of the six chromosomes constituting the R. pomonella genome (we had no marker on the small dot sixth chromosome). Moreover, six of these loci reside in inverted regions on chromosomes 1–3 that are in linkage disequilibrium with allozymes showing host-related differences among R. pomonella taxa (Roethele et al., 2001;Feder et al., 2003b). These rearranged regions contain genes affecting diapause life-history variation that are responsible for generating reproductive isolation between the sibling species and host races (Feder et al., 1997a, b; Filchak et al., 2000). As such, these six sequenced loci are located in what could be considered ‘islands of speciation’ for R. pomonella and are prime targets for reflecting population divergence. Nevertheless, moving from the temporally flat, infinite-allele framework of the allozymes to the genealogically deeper, infinite-site perspective provided by the current DNA sequence data did not appreciably change the resulting view of the R. pomonella in-group taxa. It is conceivable that advances in molecular techniques will soon make it possible to cost effectively sequence a large portion of the genome of R. pomonella for variation and reveal an essential core of fixed, host-related substitutions defining the different species. However, we must also entertain the possibility that even more exhaustive searching of the in-group taxa will not greatly change the implications of this study; R. pomonella host races and sibling species represent a seamless transition that form without the complete closure of the genome to gene flow and in the absence of the evolution of fixed genetic differences. Only when we move to the level of R. cornivora at the base of the R. pomonella complex where nearly complete pre- and post-zygotic isolations are present (Berlocher, 2000) do we observe reciprocally discreet genetic differences throughout the genome.
Speciation mode plurality
With respect to the second issue of speciation mode plurality, the sequence data are consistent with the hypothesis that past geographic isolation and subsequent introgression of inversion polymorphism from an isolated hawthorn-infesting fly population of R. pomonella in the EVTM of Mexico played a role in the adaptive radiation of the USA in-group taxa (Feder et al., 2003a). From this study, we could infer that the initial isolation of the EVTM population in Mexico (estimated at ∼1.57 Ma based on a mtDNA insect molecular clock; Feder et al., 2003a) occurred after the divergence of R. cornivora from the rest of the complex. Moreover, the introgression of inversions from the EVTM into the USA appears to have preceded the genesis of R. mendax, R. zephyria and the flowering dogwood fly, as well as the apple race. Additional information is needed, however, to assess the current taxonomic status of EVTM flies. Hawthorn-infesting populations of R. pomonella in the EVTM of Mexico clearly formed a monophyletic clade from the rest of the group for several loci. The EVTM population also does not presently appear to be in contact with hawthorn-infesting populations in the SMO or USA (Rull et al., 2006). The genetics and biogeography of EVTM population are therefore consistent with these flies currently being completely reproductively isolated from the other R. pomonella taxa and potentially having been formed via an allopatric mode of speciation not involving host shifting. However, further studies involving crosses of EVTM to SMO and US flies are needed to test for nonhost-related reproductive isolation to confirm this hypothesis.
The status of hawthorn flies in the SMO of Mexico is also not definitive. The SMO population contains genetic elements from both the EVTM and the USA, consistent with it having served as a conduit for past gene flow connecting the EVTM with an ancestral US population of hawthorn-infesting flies. Incomplete lineage sorting in combination with low levels of recombination in inverted regions of chromosomes 1–3 could contribute to the mosaic nature of the SMO gene pool, but this explanation alone is insufficient to account for the totality of the pattern. In the neighbour-joining networks (Fig. 3), the alleles in the SMO showing affinity to SN haplotypes in the USA are more closely related to each other than are those genes in the SMO and EVTM showing affinity. In addition, mtDNA indicates a close genetic relationship between SMO and US R. pomonella populations (Fig. 2d). These data imply a temporal dynamic of differential introgression, with more extensive and recent gene flow between the USA and the SMO than between the SMO and EVTM. Indeed, it is not obvious whether the differences seen between SMO and US hawthorn fly populations are indicative of interspecific divergence or conspecific geographic variation. Tentative support for the latter hypothesis comes from a recent survey of microsatellites in SMO and US hawthorn flies revealing clinal variation for several loci between the regions (Michel et al., 2007). More extensive genetic surveys and tests for nonhost-related reproductive isolation are needed to clarify the taxonomic status of SMO flies.
Patterns of genetic differentiation across the genome
Our third goal was to assess patterns of genetic differentiation across the R. pomonella genome. In this regard, with the exception of P1700, there was a general trend for loci residing on chromosomes 1–3 to display more pronounced levels of allelic divergence within and among populations than genes mapping to chromosomes 4 and 5. Previous studies have documented this pattern among EVTM, SMO and US hawthorn-infesting populations of R. pomonella (Feder et al., 2005; Xie et al., 2007). Here, we observe that the same pattern holds for the in-group taxa R. mendax, R. zephyria and the flowering dogwood fly. The result implies that the rearranged regions of chromosomes 1–3 have become less permeable to gene flow among fly taxa than loci in putative co-linear regions of the genome, consistent with recent inversion models for speciation (Noor et al., 2001a; Rieseberg, 2001; Navarro & Barton, 2003; Kirkpatrick & Barton, 2006). Against this backdrop, the lack of a signature of mtDNA introgression between the EVTM and SMO/US populations since the initial isolation of Mexican highland flies ∼1.57 Ma is puzzling. Possible explanations for the disjunct nature of mtDNA variation between the EVTM and SMO/USA include male-driven gene flow, cytonuclear gene incompatibilities, and/or a factor directly under differential selection in the mtDNA.
A delta of life?
In conclusion, our studies of the R. pomonella complex suggest that many of the dichotomies we impose concerning geographic modes/mechanisms of divergence, the cladistic splitting of taxa and systematic categories of organisms may blur during speciation. Rather than the analogy of a branching ‘tree of life’, a ‘delta of life’ comprised of many inter-tangled channels may be more appropriate for describing the formative stages of R. pomonella speciation. Thus, a plurality of mechanisms and processes including host-associated selection, sympatric host shifts, biogeography, secondary contact, past differential introgression, inversions, clines and ongoing and low-level hybridization probably interact to generate what we call species in the R. pomonella complex. These species do not necessarily display reciprocal monophyly at their inception. Rather, they exist as genotypic clusters that can be statistically identified on the basis of near private alleles and frequency differences at a subset of loci resulting from differential selection and gene flow barriers across the genome. Thus, we see no obvious qualitative genetic difference between the apple and hawthorn host races of R. pomonella, R. mendax, R. zephyria or the flowering dogwood fly in the current sequence-based analysis or past allozyme studies. Eventually, some of these species may continue to be channelled by divergent ecological selection alone or in combination with processes acting during periods of geographic isolation to consolidate into the types of discreet operational taxonomic units (OTUs) favoured by systematists for classification. Such a scenario may help explain the monophyly of R. cornivora and the EVTM population of hawthorn flies. However, care must be taken in interpreting phylogenies of such OTUs, as during the formative period of population divergence, there may be no one single bifurcating population history encapsulating the genetic and biogeographic/ecological processes that acted across the genome to generate these ‘species’. By contrast, other taxa may persist for extended periods of time in a dynamic equilibrium of semi-permanent divergence dictated by the balance between disruptive selection and introgression, whereas still others may fuse if environmental/ecological circumstances were to change. This view of life and the genesis of biodiversity is by no means unique to Rhagoletis (e.g. Wang et al., 1997; Rieseberg et al., 1999; Noor et al., 2001b; Beltrán et al., 2002; Machado & Hey, 2003; Stump et al., 2005; Turner et al., 2005; Mallet et al., 2007). It will be interesting to see whether and how this view changes as the entire genome sequences of more and more of life’s diversity become known.
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The authors thank the following individuals for their assistance: S. Berlocher, G. Bush, B. Matta, B. McPheron, J.J Smith, F. Wang, F. Wang Jr, J. Wise and the Trevor Nichols Research Station of Michigan State University at Fennville, MI. This research was supported, in part, by grants from the National Science Foundation, a National Research Initiative grant from the United States Department of Agriculture, and the 21st Century Fund of the state of Indiana to JLF.
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Table S1 Collecting sites for flies genetically analysed in the study.
Figure S1 Parsimony gene tree for locus P181.
Figure S2 Parsimony gene tree for locus P667.
Figure S3 Parsimony gene tree for locus P7.
Figure S4 Parsimony gene tree for locus P661.
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