Molecular Phylogenetics and Evolution 48 (2008) 240–257

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Molecular Phylogenetics and Evolution

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Phylogenetic analysis of nuclear and mitochondrial genes reveals evolutionaryrelationships and mitochondrial introgression in the sertifer species groupof the genus Neodiprion (Hymenoptera: Diprionidae)

Catherine R. Linnen *, Brian D. FarrellDepartment of Organismic and Evolutionary Biology and Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA

a r t i c l e i n f o

Article history:Received 14 November 2007Revised 16 February 2008Accepted 12 March 2008Available online 21 March 2008

Keywords:DiprionidaeConifer sawfliesHost useHybridizationSpeciationHypothesis testing

1055-7903/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ympev.2008.03.021

* Corresponding author. Fax: +1 617 495 5667.E-mail address: clinnen@oeb.harvard.edu (C.R. Lin

a b s t r a c t

Neodiprion Rohwer (Hymenoptera: Diprionidae) is a Holarctic genus of conifer-feeding sawflies with aremarkable amount of inter- and intraspecific diversity in host use, behavior, and development. This var-iation is thought to play a central role in Neodiprion diversification, but speciation hypotheses remainuntested due to a lack of a robust phylogenetic estimate. Here, we utilize sequence data from threenuclear genes (CAD, ANL43, EF1a) to obtain a phylogenetic estimate for the genus. These analyses suggestthat: (1) North American and Eurasian Neodiprion are monophyletic sister clades, (2) the sertifer group isparaphyletic with respect to the monophyletic lecontei group, and (3) on at least two occasions, dispersalfrom eastern to western North America proceeded via southern host bridges. Based on these results andhost biogeography, we revise a previous scenario for the evolution of Neodiprion and suggest maximumages for the genus and for the lecontei group (25 My and 14 My, respectively). In addition, because a pre-vious study reported rampant mitochondrial introgression in the lecontei group, we assess its prevalencein the sertifer group. Analysis of three mitochondrial genes (COI, tRNA-leucine, and COII) reveals thatmito-nuclear discordance is prevalent in the sertifer group, and patterns of species monophyly are con-sistent with those expected under frequent mitochondrial introgression. As was the case for leconteigroup species, we find that introgression appears to be most pronounced between species that occasion-ally share hosts, suggesting that divergent host use is an important barrier to gene flow in Neodiprion.Finally, we suggest that the lack of phylogenetic resolution and prevalence of species non-monophylyin the non-Pinus feeding Neodiprion may result from the rapid divergence (possibly with gene flow) ofthese species following their entry into a novel adaptive zone.

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1. Introduction

With over 50 described species, subspecies, and species com-plexes, Neodiprion Rohwer is the most diverse genus in the conifersawfly family Diprionidae (Hymenoptera, Symphyta). Neodiprionlarvae feed exclusively on host plants in the family Pinaceae, andbecause many species are pests of valuable timber species (Smith,1979; Arnett, 1993), Neodiprion life histories have been extensivelydocumented. These investigations have revealed a remarkableamount of intra- and interspecific variation in host use, behavior,and development (reviewed in Ross, 1955; Coppel and Benjamin,1965; Knerer and Atwood, 1973; Knerer, 1993), and this variationis thought to have played a critical role in Neodiprion diversifica-tion (Knerer and Atwood, 1973; Knerer, 1991). However, despitemuch speculation regarding Neodiprion speciation modes (e.g.,Ross, 1955; Ghent and Wallace, 1958; Alexander and Bigelow,1960; Knerer and Atwood, 1972, 1973; Bush, 1975a,b; Tauber

ll rights reserved.

nen).

and Tauber, 1981; Strong et al., 1984), we currently lack the robustphylogenetic estimate needed to evaluate previous ecological andgeographical hypotheses.

A single phylogenetic hypothesis has been proposed for thegenus Neodiprion: in his 1955 revision of the genus, Ross includeda phylogeny for 28 Neodiprion species that was supported by ahandful of morphological and ecological synapomorphies (Fig. 1).Based on this topology and changes in the historical distributionsof host plants, Ross hypothesized that Neodiprion originated inand spread across North America during the mid-Tertiary and di-verged into eastern and western lineages (the lecontei and sertiferspecies groups, respectively) following the formation of the GreatPlains region. To explain the anomalous distributions of two trans-continental sertifer group species (which are morphologically dis-tinguishable from lecontei group species), Ross hypothesizedrange expansions via northern host bridges that connected easternand western North America during the Pleistocene. In addition,Ross suggested that the ancestor to N. sertifer, the only Eurasianspecies known at the time, originated in western North Americaand achieved its present day European distribution via a dispersal

Fig. 1. Neodiprion relationships suggested by Ross (1955). Shared morphological and ecological characters are given for hypothesized clades (‘‘lancet,” ‘‘annuli”, and ‘‘ventrallobe” all refer to characteristics of the female ovipositor, or saw). Republished with permission of The Society of American Foresters, from ‘‘The taxonomy and evolution of thesawfly genus Neodiprion” by H.H. Ross in Forest Science, volume 1, issue 3, 1955; permission conveyed through Copyright Clearance Center, Inc.

C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257 241

through eastern Asia (this species was introduced into easternNorth America in 1925; Schaffner, 1939).

Thirty years after Ross published his hypothesis, six new sertifergroup species were described from China (Xiao et al., 1985). Inaddition, a recent molecular phylogenetic analysis that focusedon the lecontei group suggested that North American Neodiprionform a monophyletic group and that the western North Americansertifer group is paraphyletic with respect to the lecontei group(Linnen and Farrell, 2007). These findings, which support separateEurasian and North American Neodiprion radiations, conflict withRoss’s (1955) hypothesis of a monophyletic sertifer group that radi-ated in western North America before spreading through Chinainto Europe. However, a more rigorous evaluation of sertifer groupmonophyly and additional hypotheses proposed by Ross (e.g.,monophyly of non-Pinus feeding Neodiprion) requires a phyloge-netic analysis with denser taxonomic sampling of the sertifergroup.

In this paper we report on analyses of DNA sequence data fromthree mitochondrial and three nuclear genes sampled from multi-ple Neodiprion species, including exemplars for most lecontei groupspecies, European and Asian representatives of the sertifer group,and a dense sampling of the sertifer group species from westernNorth America. Previous work has shown that mitochondrial intro-gression is prevalent in Neodiprion; therefore, we use nuclear genesto estimate Neodiprion phylogeny (Linnen and Farrell, 2007). Wethen use relationships inferred from nuclear genes to: (1) testRoss’s hypotheses that the sertifer, lecontei, and non-Pinus-feedinggroups represent monophyletic clades within Neodiprion; (2) eval-uate Ross’s hypothesis that N. nanulus and N. abietis extended theirranges into eastern North America via northern host bridges; and

(3) identify groupings of populations of unidentified species toguide future taxonomic efforts in the sertifer group. Finally, be-cause mitochondrial introgression has been evaluated in the lec-ontei group only, we utilize mitochondrial data from ourexpanded sertifer sample to assess the prevalence of mitochondrialintrogression across the entire genus.

2. Materials and methods

2.1. Specimen collection and DNA sequence data

The majority of specimens included in this study were collectedin the United States and Canada in 2001–2004, primarily throughthe use of a beating sheet and/or visual searches for feeding colo-nies. Larvae were targeted so that larval coloration and feedinghabits could be associated with each collection. For each larval col-ony collected (or pooled solitary individuals from a single location),a subset of larvae was preserved in 100% ethanol and remainingindividuals were reared to adults and frozen at �80 �C upon emer-gence. Identifications were based on larvae and reared females(e.g., Atwood and Peck, 1943; Ross, 1955; Becker et al., 1966; Beck-er and Benjamin, 1967; Wilson, 1977; Knerer, 1984; Sheehan andDahlsten, 1985; Smith and Wagner, 1986; Dixon, 2004; an unpub-lished key to Ontario larvae by Lindquist, Miller, and Nystrom ofthe Great Lakes Forest Research Center in Sault Ste. Marie, Ontario;and an unpublished key to Florida larvae by H. Greenbaum, 1972).

In total, 126 Neodiprion specimens, representing an estimated40 species, were analyzed. From the sertifer group, representativesof 12 described North American species and two Eurasian species(N. sertifer and N. dailingensis) were included in molecular analyses.

Table 1PCR and sequencing primers

Locus PrimerA Sequence (50 ? 30)B NotesC

COI/COII S1859 GGAACIGGATGAACWGTTTAYCCICC* (a)COI/COII S2442B CCHACWGGAATTAAAATTTTYAGATGAYTAGC (b)COI/COII A2590 GCTCCTATTGATARWACATARTGRAAATG (c)COI/COII S3034 TAWTATGGCAGAAAAATGCR (d)COI/COII A3034 YGCATTTTTCTGCCATAWTA (d)COI/COII A3661 CCACAAATTTCTGAACATTGACCA* (e)EF1a Ef1-F GGACACAGAGATTTCATCAARAA* (d)EF1a Ef1-R TTGCAAAGCTTCRTGRTGCATTT* (d)EF1a F353 CCCAGCAGACCTACCGATAA (d)EF1a F601 AAAATGTCCCTGAAGTYCTW (d)EF1a R363 ACGCAGAGCCTTATCGGTAG (d)EF1a R601 WAGRACTTCAGGGACATTTT (d)CAD 787F GGDGTNACNACNGCNTGYTTYGARCC* (f)CAD 1098R TTNGGNAGYTGNCCNCCCAT* (f)CAD CAD-F1 ARATHGCNACHGCGGTVAARAG (d)CAD CAD-R1 TYTTBACCGCDGTNGCDATYTG (d)ANL43 43F ACAAAACGAAAGGCCAGAGG* (d)ANL43 43R GATTCCGATCAACCCCTTCA* (d)ANL43 43-SF1 GTTACTTACTTACRCAGCAK (d)ANL43 43-SF2 AACGGKTACGAAGCATAATA (d)ANL43 43-SR1 TTTTCGCKRTGCTCAAGYCG (d)ANL43 43-SR2 GTCAYCTGACCTTTATCTTG (d)ANL43 43-F49 TCCCWCTRCAAATAGAYA (d)ANL43 43-F425 TACAAGATAAAGGTCAGRTG (d)ANL43 43-R286 CTGACTAACGGMGASACAAT (d)ANL43 43-R778 GGTTTACMTCAATCCYTCTG (d)

A Letters refer to primer direction (S, sense; A, antisense; F, forward; R, reverse).For COI/COII, numbers refer to position of the primer in the Drosophila yakubamtDNA genome (Clary and Wolstenholme, 1985; Simon et al., 1994). For theremaining loci, numbers are either arbitrary (as is the case for CAD-F1, CAD-R1, 43-SF1, 43-SF2, 43-SR1, and 43-SR2) or refer to relative annealing locations ofsequencing primers.

B *, primer used for both sequencing and PCR; no asterisk, primer used forsequencing only.

C Notes and references: (a) C1-J-1859 from Simon et al. (1994); (b) Modified fromNormark et al. (1999) by B. O’Meara; (c) Normark et al. (1999); (d) Designed for thisstudy; (e) C2-N-3661 from Simon et al. (1994); (f) From Moulton and Wiegmann(2004).

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In addition, many individuals that cannot be confidently identifiedat this time were collected. These individuals most likely representa combination of undescribed species and members of morpholog-ically-confusing species complexes (Ross, 1955) and are includedin the phylogenetic analyses described here to obtain a more com-plete picture of relationships within the sertifer group and to in-form future taxonomic work. When possible, multiplepopulations were included for sertifer group species (described orundescribed ‘‘morpho-species”) so that species boundaries couldbe evaluated. From the lecontei group, exemplars of 17 of the 19known species and two recently discovered species (Linnen andFarrell, 2007; Linnen, 2007) were included in phylogenetic analy-ses. Finally, a species belonging to the closely related genus Zadip-rion Rohwer was included as an outgroup. Zadiprion was originallydesignated as a subgenus within Neodiprion by Rohwer (Rohwer,1918; Ross, 1937), then given generic status by Ross (1937). Thisstatus change is supported by mitochondrial and nuclear sequencedata, which recover Neodiprion and Zadiprion as monophyletic sis-ter genera within Diprionidae (Linnen, unpublished data). All spec-imens included in this study are listed in Appendix A withassociated collecting data and GenBank accession numbers.

Sequence data was obtained for each of the 127 specimens (126Neodiprion and 1 outgroup) included in this study from the follow-ing gene regions: a large region spanning the mitochondrial genescytochrome c oxidase I, tRNA-leucine, and cytochrome c oxidase II(COI/COII), a region of the F2 copy of elongation factor-1a (EF1a thatspanned portions of two exons and a large intervening intron(Danforth and Ji, 1998; Danforth et al., 1999; Nyman et al.,2006); a region of rudimentary (CAD) that spanned portions oftwo exons and two introns; and an anonymous nuclear locus(ANL43). For the lecontei group species and some sertifer groupspecies, this data was available from a previous study (Linnen andFarrell, 2007; GenBank Accession Nos. EF361837–EF362376). Forall remaining specimens, DNA was extracted, amplified, andsequenced as described in Linnen and Farrell (2007) using theprimers listed in Table 1.

Sequencher version 4.1 (GeneCodes, Ann Arbor, MI) was used toassemble contigs, confirm base calls (by eye), and to confirm thatall protein-coding regions had open reading frames. Only a singlebase pair deletion was present in the tRNA-leucine gene of sometaxa; therefore, the mitochondrial gene region (COI/COII) was eas-ily and unambiguously aligned by eye. The three nuclear regionswere aligned using default settings in Clustal X version 1.83(Thompson et al., 1997), followed by a manual adjustment inMacClade version 4.05 (Maddison and Maddison, 2000). EF1a andCAD exons did not contain any insertions or deletions, and the in-trons of these genes contained few gaps, all of which were easilyaligned. In contrast, ANL43 had a large repetitive region that washighly polymorphic in length and could not be unambiguouslyaligned—this region was trimmed from the dataset before furtheranalysis. The final aligned dataset for all genes and all individualswas 4562 base pairs (bp) in length, (1752 bp COI/COII, 1104 bpEf1a, 911 bp CAD, and 795 bp ANL43). Sequences that were not pre-viously published in Linnen and Farrell (2007) were submitted toGenBank (Accession Nos. EU279461–EU279852; see Appendix A).

2.2. Phylogenetic analysis

Bayesian, Maximum Likelihood (ML), and Maximum Parsimony(MP) analyses were carried out on concatenated nuclear data be-cause previous analyses suggest that the three nuclear genes(Ef1a, CAD, and ANL43) share an underlying history (the Neodiprion‘‘species tree”, Linnen and Farrell, 2007) and because this approachis expected to maximize phylogenetic signal in the data (Kluge,1989; de Queiroz et al., 1995; Baker and DeSalle, 1997; Wiens,1998; Lerat et al., 2003; de Queiroz and Gatesy, 2007). Mitochon-

drial genes were analyzed separately because they introgress read-ily in Neodiprion and therefore do not appear to share the samehistory as nuclear genes (Linnen and Farrell, 2007).

A Bayesian analysis was performed in MrBayes version 3.1(Ronquist and Huelsenbeck, 2003) with the following models cho-sen according to the Akaike information criterion (AIC) andMrModeltest version 2.2 (Nylander et al., 2004): HKY + C (EF1a),GTR + C (CAD), and GTR + I + C (ANL43). The nuclear analysis waspartitioned by gene (ANL43, EF1a, and CAD), model parameterswere unlinked across data partitions, and among-partition ratevariation was accommodated using rate multipliers (option: prsetratepr=variable; see Marshall et al., 2006). To investigate theimpact of partition choice on Bayesian analyses, two additional par-titioning schemes were evaluated: (1) a five partition scheme inwhich protein-coding genes (EF1a and CAD) were partitioned intointrons and exons, and (2) a seven partition scheme in which exonswere further divided into first + second, and third codon positions.For these partitions, the following models were chosen using theAIC and MrModeltest: GTR + C (CAD exons), HKY + I (CAD exon 1stand 2nd position), HKY (CAD exon 3rd position; CAD introns),SVM + C (EF1a exons), F81 (EF1a exon 1st and 2nd position); K80(EF1a exon 3rd position); GTR (EF1a intron). Finally, for comparisonwith results obtained from concatenated nuclear data, a separateBayesian search was performed for each nuclear gene using modelschosen by the AIC and MrModeltest. Areas of conflict and congru-ence between nuclear genes were visualized using a consensus net-work (Holland and Moulton, 2003) constructed in SplitsTree 4version 4.6 (Huson and Bryant, 2006). All Bayesian analyses con-sisted of two concurrent runs, four Markov chains (one cold chain

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and three heated chains, with a temperature of 0.1), 10 million gen-erations (sampled every 1000 generations), and a 25% burn-in. Runswere considered to have converged on the stationary distributionwhen there were no obvious trends in generation vs. log-likelihoodplots and potential scale reduction factors (PRSF) were near 1.0 forall parameters (Ronquist et al., 2005).

To evaluate the consistency of results across analysis methods,ML and MP searches were performed on the concatenated nucleardata. ML searches were performed in GARLI version 0.951 (Zwickl,2006) using a model chosen by MrModeltest and the AIC(GTR + I + C was chosen for the concatenated nuclear dataset).Automatic termination was enforced and runs were stopped when250,000 generations had passed without a significantly better scor-ing topology. To check for consistency of topologies and likelihoodscores, multiple runs were performed. When similar topologiesand likelihood scores (all within one log-likelihood unit) were ob-tained from three independent runs, the topology with the bestlikelihood score of these three was selected. A bootstrap analysiswas also performed in GARLI and consisted of 500 replicates withthe automatic termination criterion reduced to 5000 generations.An MP search was performed in PAUP* 4.0b10 (Swofford, 2000)and consisted of 1000 random addition sequences (RAS), treebisection-reconnection (TBR) branch swapping, and no more than10 trees saved per RAS to reduce computation time. MP resultswere summarized using a strict consensus. An MP bootstrap searchconsisted of 1000 replicates, each with 10 RAS, TBR branch swap-ping, and no more than 10 trees saved per RAS.

2.3. Testing Ross’s 1955 hypotheses

Monophyly hypotheses were evaluated for the following threeclades proposed by Ross (1955): (1) the sertifer species group, (2)the lecontei species group, and (3) non-Pinus feeding Neodiprion.In addition, because Asian sertifer species were unknown to Rossin 1955, three possibilities for sertifer monophyly were considered:(1) all sertifer group species form a monophyletic group (Fig. 2A),(2) European and North American sertifer form a monophyleticgroup (Fig. 2B), and (3) only North American sertifer form a mono-phyletic group (Fig. 2C).

All hypotheses were evaluated using nuclear data and both like-lihood and Bayesian approaches. First, each monophyly hypothesiswas tested in a likelihood framework by asking whether theunconstrained ML tree fit the data significantly better than themost likely tree that conformed to that hypothesis—if the uncon-strained tree was significantly more likely, that monophylyhypothesis was rejected. Constraint trees corresponding to eachmonophyly hypothesis were constructed in MacClade version4.05 (Maddison and Maddison, 2000), and ML searches with theseconstraints enforced were performed in Garli version 0.951. As was

lecontei group

lecontei group

sertifer group (All)

sertifer group (Eur+NAm)

Fig. 2. Possible hypothesis for sertifer group monophyly. (A) Corresponds to Ross’s 19

the case for unconstrained searches, multiple Garli runs were per-formed for each constrained search. When three independent runsreturned scores that were all within one log-likelihood unit for agiven constraint, the tree with the best likelihood score wasselected for hypothesis testing. The significance of the differencesin likelihoods between the unconstrained and constrained topolo-gies was tested using Shimodaira–Hasegawa (SH) tests (Shimoda-ira and Hasegawa, 1999), implemented in PAUP* with the RELLapproximation and 10,000 bootstrap replicates. Second, Bayesiantests of monophyly were performed by importing each monophylyconstraint into PAUP* and filtering the post-burn-in set of trees.Hypotheses were rejected if less than 5% (1% after Bonferroni cor-rection for n = 5 tests) of the trees were retained after filtering witha given constraint tree (Miller et al., 2002; Buschbom and Barker,2006; Linnen and Farrell, 2007).

2.4. Assessment of mitochondrial introgression

For comparison with relationships inferred from concatenatednuclear data, Bayesian, ML, and MP analyses were carried out onmitochondrial (COI/COII) data; analysis methods were exactly asdescribed for the nuclear data, and the GTR + I + C model (chosenusing the AIC and MrModeltest) was used in Bayesian and ML anal-yses (see ‘‘Section 2.2” section for analysis details). In addition, toinvestigate the impact of partition choice on mitochondrial analy-ses, two additional partitioning schemes were evaluated: (1) athree partition scheme in which the mitochondrial region was par-titioned by gene (COI, tRNA-leucine, and COII), and (2) a five parti-tion scheme in which COI and COII were further divided intofirst + second, and third codon positions. For these partitions, thefollowing models were chosen using the AIC and MrModeltest:GTR + I + C (COI; COI 3rd position; COII; COII 3rd position), HKYI + C (COI 1st and 2nd position), HKY + I (COII 1st and 2nd position),HKY (tRNA-leucine). Finally, for comparison with separate mito-chondrial and nuclear analyses, a Bayesian analysis was performedon concatenated data (four partitions: EF1a, ANL43, CAD, and COI/COII)—with the exception of the run length (which was 15 milliongenerations), analysis conditions for the full dataset were identicalto separate mitochondrial and nuclear analyses.

The most direct test of biased mitochondrial introgressionwould be to compare introgression rates across the four gene re-gions; however, species boundaries in the sertifer group have notbeen adequately characterized to perform such tests (Linnen andFarrell, 2007). Therefore, the extent of mitochondrial introgressionwas assessed using Bayesian tests of monophyly for identified ser-tifer species. If mitochondrial introgression has been prevalent inthe sertifer group, mitochondrial gene trees are predicted to re-cover an equal or greater number of non-monophyletic speciesthan do nuclear gene trees. In the absence of introgression, mito-

lecontei group

sertifer group (Asia)

sertifer group (NAm)

sertifer group (Eurasia)

55 hypothesis, which was proposed before the discovery of Chinese Neodiprion.

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chondrial gene trees are predicted to recover more monophyleticspecies because they have a smaller effective population size, onaverage, than nuclear loci (Palumbi et al., 2001; Ballard and Whit-lock, 2004). These predictions were tested in a Bayesian frameworkbecause the posterior probabilities of trees can be interpreted asthe probability that those trees are correct (assuming correct mod-el specification; Huelsenbeck and Rannala, 2004). For these tests,11 constraint trees were constructed in MacClade version 4.05(Maddison and Maddison, 2000) to correspond to hypotheses ofmonophyly for each of the 11 identified species for which multiplepopulations had been sampled. These monophyly constraints werethen imported into PAUP* and used to filter the post-burn-in set oftrees obtained from each of six separate Bayesian analyses [ANL43,CAD, EF1a, concatenated nuclear (‘‘Nuclear”), COI/COII, and concat-enated mitochondrial and nuclear (‘‘All”)]. Hypotheses of mono-phyly were rejected if less than 5% (0.45% after Bonferronicorrection for multiple comparisons within each set of trees) ofthe trees from a given analysis were retained after filtering witha given constraint tree (Miller et al., 2002; Buschbom and Barker,2006; Linnen and Farrell, 2007). Finally, because unidentified spe-cies could potentially cause spurious rejections of monophyly (e.g.,for the species to which they should have been assigned), Bayesiantests of monophyly were repeated on a pruned dataset that con-tained only identified species (unidentified species were alsopruned from constraint trees).

3. Results

3.1. Phylogenetic analysis

Lengths, percentages of variable sites, and models chosen byMrModeltest and the AIC are given for each gene region in Table2; Fig. 3 summarizes the results obtained in Bayesian, ML, andMP analyses of the concatenated nuclear dataset. Agreement be-tween analysis methods was good, as is demonstrated by the lackof conflicting nodes—in no cases were there clades recovered in theBayesian analysis that conflicted with clades recovered in ML andMP analyses (there were, however, clades that were unresolvedin these analyses). Likewise, results were nearly identical acrossall partitioned Bayesian analyses; therefore, only the results fromthe simplest partitioning scheme (three nuclear genes) are shown.

The backbone of the Neodiprion phylogeny was well resolved bynuclear genes and there was strong support (>95% for all methods)for the monophyly of the North American Neodiprion, EurasianNeodiprion, and the lecontei group (these clades were also recov-ered with strong support when additional Zadiprion species anddiprionid genera were included as outgroups; results not shown).In addition, there was a moderate level of support (>70% for allmethods) for a single large radiation in which either a single ormultiple shifts to non-Pinus hosts occurred. Finally, phylogeneticanalysis of the concatenated nuclear data also suggests that thesertifer group is paraphyletic with respect to the lecontei groupand that there have been multiple independent colonizations ofeastern North America from western North American ancestors.

Table 2Sequence characteristics and substitution model for each locus

Locus Length (bpa) Variable sites (%) PIb Sites (%) Model

ANL43 795 25.0 10.6 GTR + I + CCAD 911 14.2 6.4 GTR + CEF1a 1104 24.3 11.2 HKY + CNuclear 2810 21.2 9.5 GTR + I + CCOI/COII 1752 31.8 23.5 GTR + I + C

a bp, base pairs.b PI, parsimony-informative.

Phylogenetic analysis of nuclear genes also supported themonophyly of several sertifer group species (e.g., N. abietis, N. tsu-gae, N. edulicolus, N. demoides, N. autumnalis, and N. ventralis).Remaining species were generally recovered as paraphyletic (e.g.,N. deleoni, N. scutellatus)—most notable among these, the Europeanspecies, N. sertifer, appears to be paraphyletic with respect to theChinese species, N. dailingensis. The only clear case of polyphylywas a group of individuals (referred to here as N. abietis 2) thathave been previously referred to as the ‘‘white fir strain” of theN. abietis species complex (Ross, 1955; Knerer and Atwood, 1972,1973; Sheehan and Dahlsten, 1985).

In general, nuclear genes did not provide much resolution forrelationships between species; this was particularly apparentwithin the group containing non-Pinus feeders. Therefore, thenumber of origins and/or losses of non-Pinus feeding cannot beevaluated at this time. This lack of resolution likely stemmed froma combination of (1) insufficient phylogenetically informative var-iation and (2) conflict between nuclear gene trees (resulting fromstochastic lineage sorting and/or introgression). The first explana-tion was supported by the observation that phylogenies estimatedfrom individual nuclear genes are poorly resolved (SupplementaryFigures 1–3). In addition, examination of the consensus networkconstructed from individual gene trees revealed that nuclear genesrecover at least some conflicting relationships (Fig. 4A).

Even though some portions of the Neodiprion tree were poorlyresolved, some groupings of interest did emerge. Most notably,N. ventralis was recovered as sister to the lecontei group; also clo-sely related were N. autumnalis, N. nr. omosus, and N. gillettei. Thesespecies are distributed throughout the southwestern US and inMexico, suggesting a possible southern origin for the leconteigroup. In addition, Arizona populations of N. nr. abietis 1 weresupported as the sister group to eastern North American N. abietis.

3.2. Testing Ross’s 1955 hypotheses

Table 3 reports results obtained from SH and Bayesian tests ofRoss’s 1955 hypotheses. First, both SH and Bayesian tests of sertifergroup monophyly strongly rejected the monophyly of all sertifergroup species (Fig. 2A) and of European + North American sertifergroup species (Fig. 2B). In contrast, the monophyly of all NorthAmerican sertifer group species could not be rejected statisticallyby either method, although the Bayesian test was only marginallynon-significant (Fig. 2C). Second, the monophyly of the leconteigroup could not be rejected because this clade was present in theML tree (i.e., D �LnL = 0); lecontei non-monophyly was marginallynon-significant according to the SH test, but was strongly rejectedby the Bayesian test. Third, results for non-Pinus feeding Neodiprionalso depended on method: the SH test did not reject the mono-phyly non-Pinus feeders, but the Bayesian test did (p = 0).

3.3. Assessment of mitochondrial introgression

Fig. 5 summarizes the results obtained in Bayesian, ML, and MPanalyses of the mitochondrial data. As was the case for the nucleardata, results for mitochondrial data were nearly identical across allpartitioning schemes; therefore, only the results from the unparti-tioned analysis are shown. Like the nuclear analyses, mitochondrialanalyses recovered the sertifer group as paraphyletic, had moderatesupport for a single radiation in which a shift to non-Pinus feedingoccurred, and strongly supported the monophyly of the NorthAmerican Neodiprion, the Eurasian Neodiprion, and the leconteigroup. Aside from these basic similarities, however, relationshipsimplied by mitochondrial and nuclear genes were very different.In contrast to nuclear genes, mitochondrial genes recovered mono-phyly for few species (e.g., N. autumnalis and N. ventralis) andpolyphyly for many (e.g., N. tsugae, N. edulicolus, N. scutellatus,

Fig. 3. Bayesian cladogram with Bayesian, likelihood, and parsimony support values from combined analysis of nuclear genes. Support is given for selected nodes in thefollowing order: Bayesian posterior probabilities (BPP)/maximum likelihood bootstrap (MLB)/maximum parsimony bootstrap (MPB). Stars indicate species for whichmonophyly was rejected statistically (see Table 4 and Fig. 5); dots indicate individuals that were collected on non-Pinus hosts; vertical bars indicate species that occur ineastern North America. Additional details are indicated in the figure legend.

C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257 245

Fig. 4. Consensus network for (A) EF1a, ANL43, and CAD gene trees (Supplementary Figures 1–3) and (B) EF1a, ANL43, CAD, and COI/COII gene trees (Supplementary Figures1–3; Fig. 5). Numbers correspond to specimen identification numbers in Appendix A. The presence of boxes in these figures indicates conflict between different datasets.

246 C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257

Table 3Shimodaira–Hasegawa and Bayesian tests of Ross’s, 1955 hypotheses

Constrainta �LnL 4 �LnL p-Valueb % Treesc

None 9495.74 n/a n/a 100Sertifer monophyly A 9526.31 30.57 0.0009 0Sertifer monophyly B 9715.85 220.11 0.0000 0Sertifer monophyly C 9502.41 6.67 0.1499 1.01Lecontei non-monophyly 9530.57 34.83 0.0194 0non-Pinus monophyly 9521.70 25.96 0.1135 0

a See Fig. 2 for explanation of sertifer monophyly A, B, and C.b Significant p-values (a = 0.01 after Bonferroni correction for n = 5 tests) are

shown in bold.c Percentage of post-burn-in trees obtained in Bayesian analysis of concatenated

nuclear data that are consistent with each hypothesis; significant values (a = 0.01)are shown in bold.

C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257 247

N. deleoni). Rampant polyphyly was particularly pronounced in thenon-Pinus feeders; the hemlock-specialist N. tsugae, for example, isspread out across two distantly related clades (whereas nucleargenes recover this species as monophyletic with moderate to highsupport, Fig. 3). Also, the sister group to the lecontei group wasunresolved in a Bayesian analysis of mitochondrial genes (andML and MP analyses each suggested different taxa).

Superficially, it appears that analysis of mitochondrial genesgives much higher resolution and support for relationships thando nuclear genes; therefore, combining all genes could have re-sulted in a well-resolved and well-supported phylogeny. This cer-tainly appears to be the case when one examines the resultsobtained from analysis of the full dataset (Supplementary Figure4). However, as would be predicted if mitochondrial genes wererecovering a different (non-phylogenetic) history from nucleargenes, there was a substantial amount of conflict between mito-chondrial and nuclear gene trees (Fig. 4B, see Fig. 4A forcomparison).

Turning to Bayesian tests of monophyly, null hypotheses ofmonophyly were rejected by each of the six data partitions; how-ever, when all specimens that could not be identified were ex-cluded from analysis, the combined nuclear partition did notreject monophyly for any of the 11 species examined (see Table4 and Fig. 6; in addition, species for which monophyly was rejectedwhen all samples were considered are indicated by stars in Figs. 3and 5). Notably, when mitochondrial data was added to nucleardata (ALL), monophyly was rejected for 5 species (3 species whenunidentified specimens are excluded)—this observation furtherdemonstrates that there was conflict between mitochondrial andnuclear datasets. With the exception of CAD, each nuclear partitionrejected species monophyly in fewer instances than did the mito-chondrial partition (when both identified and unidentified sampleswere considered, monophyly was rejected for 1 species for EF1a, 6species for ANL43, and 1 species for the combined nuclear dataset).For both CAD and COI/COII, monophyly was rejected for a majorityof the species (8 of 11 and 7 of 11, respectively). Finally, while nomonophyly hypotheses were rejected by all five data partitions,there were two species (N. autumnalis and N. ventralis) that werestrongly supported (>98% posterior probability) by all datapartitions.

4. Discussion

4.1. Evolution of the genus Neodiprion

The phylogenetic analyses presented here, along with the dis-covery of Asian Neodiprion species (Xiao et al., 1984a,b, 1985)and new information regarding Pinus biogeography (reviewed inMacDonald et al., 1998; Millar, 1998; Perry et al., 1998; Graham,1999), necessitate a revision of some (but not all) aspects of Ross’s

1955 scenario for the evolution of Neodiprion (see Fig. 1). First,Ross’s (1955) suggestion that Neodiprion originated in North Amer-ica is neither supported nor rejected by currently available data, asNorth American and Eurasian Neodiprion form strongly supportedmonophyletic sister groups. Second, the monophyly of non-Pinusfeeding Neodiprion also could not be evaluated at this time dueto a lack a resolution and support for relationships within the cladecontaining non-Pinus feeders. Third, Ross’s hypothesis that the ser-tifer and lecontei groups form monophyletic sister clades is not sup-ported—all analyses recovered the western North American sertifergroup as paraphyletic to a more recently derived, monophyleticlecontei group (generally with very high support).

Fourth, these results are consistent with Ross’s suggestion of amid-Tertiary (possibly Miocene) origin. Specifically, if the relation-ships recovered here are correct, Neodiprion was present in wes-tern North America and Asia before the ancestor to the leconteigroup colonized eastern North America; thus, the genus Neodiprionmost likely originated during a time when (1) the distribution ofPinus connected western North America and Asia and (2) therewere barriers to dispersal between eastern and western NorthAmerica. The first condition was met starting in the Oligocene(35 million years ago (Myr)), when climatic cooling promotedthe spread of pines out of Eocene refugia (Millar, 1998) and dis-persal between North America and Asia would have been possiblevia the Beringian Land Bridge (Sanmartin et al., 2001). However, anabsence of barriers to dispersal between eastern and westernNorth America from 30 to 25 Myr (Sanmartin et al., 2001) suggestsan upper age limit of 25 Myr for the genus. In addition, the exis-tence of a continuous coniferous forest connecting North Americaand Asia from the middle-late Miocene (14–10 Myr) through thelate Pliocene (3.5 Myr) (Sanmartin et al. 2001) is suggestive of amore recent Neodiprion origin because one might expect the diver-gence between Eurasian and Western North American Neodiprionto correspond to a time when barriers to dispersal were present.

The fifth (and final) portion of Ross’s evolutionary scenario thatwe examined dealt with dispersals into eastern North America.Specifically, Ross hypothesized that the current trans-continentaldistribution of N. nanulus (which he divided into eastern and wes-tern subspecies) and N. abietis originated during the Pleistocene vianorthern host bridges. This hypothesis cannot be evaluated forN. nanulus due to insufficient sampling of the western subspecies(N. nanulus contortae). In contrast, a wide geographic samplingwas obtained for western members of the N. abietis species com-plex; surprisingly, the populations that were genetically most sim-ilar to eastern N. abietis were from Arizona. This suggests thatN. abietis colonized eastern North America via a southern hostbridge during Pleistocene glaciations—such a bridge may haveformed as northern temperate elements migrated southwards intoMexico along the Sierra Madre Occidental (in the west) and theSierra Madre Oriental (in the east), which are connected by theTransvolcanic Belt in central-southern Mexico (Dressler, 1954;Perry et al., 1998). This same migration route may have allowedthe ancestor to the lecontei group to colonize eastern NorthAmerica during the Tertiary (Martin and Harrell, 1957; Perryet al., 1998). In support of this hypothesis, the lecontei groupappears to have originated from within a clade of southwesternand Mexican sertifer species and one lecontei group species (N. exc-itans) is known to occur in Central America. A recent analysis ofpalynofloras of southern Mexico and Central America suggests thatnorthern temperate trees (e.g., Pinus, Picea, Abies, and others) wererare or absent until the middle-late Miocene (�14–10 Myr; at thistime a drastic decline in global temperature occurred) and becameincreasingly common in the Pliocene and Pleistocene. Thismigration scenario therefore suggests a maximum age of 14 Myrfor the origin of the lecontei group (before this time, dispersal intoeastern North America would have been unlikely).

Fig. 5. Bayesian cladogram with Bayesian, likelihood, and parsimony support values for mitochondrial genes. Support is given for selected nodes in the following order:Bayesian posterior probabilities (BPP)/maximum likelihood bootstrap (MLB)/maximum parsimony bootstrap (MPB). Stars indicate species for which monophyly was rejectedstatistically (see Table 4 and Fig. 3); dots indicate individuals that were collected on non-Pinus hosts; vertical bars indicate species that occur in eastern North America.Additional details are indicated in the figure legend.

248 C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257

Table 4Bayesian tests of monophyly for each data partitiona

Species ANL43 CAD EF1a Nuclear COI/COII ALL

N. abietis 9.6 (10.8) 0 (0.03) 93.7 (100) 98.8 (100) 0 (0) 100 (100)N. autumnalis 99.8 (99.8) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100)N. deleoni 0 (0.01) 0 (0) 26.5 (37.7) 0.29 (97.4) 0 (0) 0 (99.9)N. demoides 1.7 (94.1) 0.01 (0.07) 100 (100) 100 (100) 0 (27.6) 99.3 (100)N. edulicolus 0.01 (0.01) 0 (0) 7.2 (100) 10.3 (100) 0 (0) 0 (0)N. mundus 0.02 (100) 0 (91.7) 99.5 (99.6) 11.1 (100) 12.0 (100) 0 (100)N. nanulus 0.05 (0.25) 0.87 (9.4) 1.9 (96.9) 29.9 (99.3) 0 (0) 0 (0)N. scutellatus 0 (5.4) 0 (0) 92.0 (100) 6.3 (100) 0 (0) 75.2 (100)N. sertifer 57.5 (57.5) 0 (0) 0.21 (0.21) 6.0 (6.0) 11.7 (11.7) 12.0 (12.0)N. tsugae 0.01 (0.32) 0 (0) 92.4 (99.8) 99.9 (100) 0 (0) 0 (0)N. ventralis 100 (100) 100 (100) 100 (100) 100 (100) 100 (100) 100 (100)Total # rejected 6 (4) 8 (7) 1 (1) 1 (0) 7 (6) 5 (3)

a For each species, the percentage of trees in the posterior probability distribution that recovered the monophyly of that species is given for each data partition. Values inparentheses correspond to percentages obtained when all unidentified species were removed from the dataset; remaining values correspond to tests carried out with allsampled individuals. Significant p-values (a = 0.0045, or 0.45%, after Bonferroni correction for n = 11 tests per dataset) are shown in bold. The total number of species forwhich monophyly was rejected is given for each data partition.

0

10

20

30

40

50

60

70

80

90

100

N. abie

tis

N. autu

mnalis

N. dele

oni

N. dem

oides

N. edu

licolus

N. mun

dus

N. nan

ulus

N. scu

tellat

us

N. sert

ifer

N. tsug

ae

N. ven

tralis

Species

Nuclear (All)Nuclear (Identified)COI/COII (All)COI/COII (Identified)

% T

rees

reco

verin

g m

onop

hyly

** ** ** ** ** *** *

Fig. 6. Percentage of trees that recovered monophyly for each of 11 sertifer group species for nuclear and mitochondrial data partitions. Results are given for both pruned(‘‘Identified”, meaning that all unidentified specimens were pruned from trees prior to monophyly tests) and unpruned (‘‘All”) datasets. Significant results (a = 0.0045 aftercorrection for multiple tests) are indicated by asterisks.

C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257 249

The evolutionary scenario proposed here updates someaspects of Ross’s hypothesis; however, much work remains tobe done. For example, additional data will be required to testthe hypothesis there has been a single shift onto non-Pinus hosts(and no losses of this trait)—at present, the evidence for thishypothesis is mixed (Bayesian methods reject non-Pinus mono-phyly, SH tests do not). In addition, while the scenario we pro-pose is based on relationships shown in Fig. 3, SH andBayesian tests were unable to reject monophyly for the westernNorth American sertifer group and the non-monophyly of the lec-ontei group was rejected by Bayesian methods, but the SH test

was marginally non-significant. Therefore, these interpretationsshould be reevaluated as new data and taxa become available.Additional sampling/study of the Eurasian clade is also needed,but perhaps the biggest gap in current knowledge exists for spe-cies in Mexico and Central America. Neodiprion diversity in thisarea is virtually unexplored, but could be quite high given theincredible diversity of pines in the region (Smith, 1988). Samplesfrom this region would also bear directly on the dispersal routeshypothesized here. Finally, analyses at the family level andmolecular dating analyses will provide additional insight intothe timing and location of Neodiprion origins.

250 C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257

4.2. Species boundaries in the sertifer group

Species boundaries suggested by phylogenetic analysis of con-catenated nuclear data (Fig. 3) generally agreed with morphology.More specifically, species that could be identified from publisheddescriptions (e.g., Ross, 1955; Sheehan and Dahlsten, 1985; Smithand Wagner, 1986) were most often found to be monophyletic orparaphyletic (which is to be expected if they are recently derived).One exception to this general pattern was that a group of individ-uals identified as members of the ‘‘white fir strain” of the N. abietisspecies complex was polyphyletic (Knerer and Atwood 1972, 1973;Sheehan and Dahlsten, 1985; referred to as N. nr. abietis 2 in Figs. 3and 5). This finding was unexpected because larvae and adultswere uniform and very distinct from all other species (final instarlarvae and eggs that were visible within the abdomen of the adultfemales were bright lime green in color; also, cocoons were nearlytranslucent). Three possible explanations for the observed nuclearpolyphyly are: (1) nuclear gene flow at the loci sampled has beenextensive, (2) this species is of hybrid origin, or (3) this species isof recent origin and polyphyly is the result of incomplete lineagesorting. Further sampling (of genes and individuals) will be re-quired to distinguish between these alternatives.

Numerous individuals belonging to the sertifer group could not beassigned a name at this time, and the majority of these were col-lected on ponderosa pine—as Ross lamented in 1955, ‘‘collectionsfrom Pinus ponderosa provide the most puzzling question in this gen-us.” Several species on this host are distinct and easily identified bylarval and adult characters (N. mundus, N. autumnalis, N. ventralis,and N. gillettei) and, in some cases, rigid preferences for particularhost age/size classes (Haack and Mattson, 1993; for example, N. gil-lettei prefers host <60 cm tall; Dunbar and Wagner, 1992). Much ofthe remaining diversity on ponderosa pine was first lumped intothe N. fulviceps complex by Ross (1955), then the N. autumnalis com-plex by Smith and Wagner (1986). In this study, female morphologyplaces most of the unidentified ponderosa-pine feeders into the ful-viceps/autumnalis complex (Smith, personal communication). How-ever, nuclear genes recovered 3–4 distinct clusters of theseindividuals (denoted by triangles in Fig. 3), and some clusters appearto be associated with consistent differences in larval coloration;these findings can be used to guide future studies of this complex.

4.3. Mitochondrial introgression

As was observed for the lecontei group (Linnen and Farrell,2007), separate analysis of mitochondrial and nuclear genes re-vealed mito-nuclear discordance in the sertifer group. While inter-specific gene flow was not measured in this case, patterns ofdiscordance in the sertifer group are reminiscent of those in the lec-ontei group and are consistent with mitochondrial introgression.Specifically, with the exception of CAD, all nuclear data partitionsrejected monophyly for fewer species than the mitochondrial par-tition. If discordance were caused by incomplete lineage sorting,one would expect the opposite pattern (i.e., more monophyleticspecies recovered by mitochondrial genes) due to the lower effec-tive population size, on average, of mitochondrial genes comparedto nuclear genes (Palumbi et al., 2001; Funk and Omland, 2003;Ballard and Whitlock, 2004). One possible explanation for CAD’sfailure to recover species monophyly is a relative lack of variation(Table 2, Supplementary Figure 3). In contrast, lack of resolutiondoes not adequately explain COI/COII’s tendency to reject speciesmonophyly because this partition had the highest percentage ofvariable and parsimony-informative characters (Table 2). Thus,we conclude that the patterns observed in the sertifer group aremost likely the result of mitochondrial introgression. However,the most direct test of mitochondrial introgression—comparisonof mitochondrial and nuclear gene flow rates—was not performed

and this conclusion should be re-evaluated when species bound-aries are sufficiently resolved to estimate interspecific gene flow.

Also in agreement with patterns observed in the lecontei group,the most extensive mitochondrial introgression (inferred in thiscase from mitochondrial polyphyly) appears to have occurred be-tween species that at least sometimes share host plants. Specifi-cally, non-Pinus feeders seem particularly prone to mitochondrialintrogression (Fig. 5 and Table 4). While some of these species spe-cialize on different genera (e.g., N. scutellatus on Pseudotsuga;N. tsugae on Tsuga; N. deleoni on Abies), members of the enigmaticN. abietis ‘‘species complex” have been reported on hosts in multi-ple genera (Pseudotsuga, Tsuga, Picea, and Abies; Ross, 1955; Knererand Atwood, 1972, 1973; Smith 1979). In addition, some of thenon-Pinus feeding species have outbreak population dynamics(particularly N. tsugae and N. abietis complex; Larsson et al.,1993) and have been reported on non-preferred hosts during out-breaks. If female mate choice is frequency-dependent, outbreakingmay also promote biased mitochondrial introgression by causingnumerical imbalance between potentially hybridizing species pairs(Linnen and Farrell, 2007; see Chan and Levin, 2005). An alterna-tive explanation that is often invoked to explain mitochondrialintrogression is female-biased dispersal. However, female-biaseddispersal is unlikely to explain widespread mitochondrial intro-gression in Neodiprion because females, which emerge from theircocoons laden with a full complement of eggs, are poor fliers anddispersal is generally male-biased (Coppel and Benjamin, 1965).

Finally, it is worth noting that monophyly for non-Pinus feederswas also frequently rejected by individual nuclear genes (Table 4)and that the clade containing non-Pinus feeders was poorly resolvedby combined analysis of nuclear genes (Fig. 3). These patterns, whichare expected under both rapid radiation and divergence-with-geneflow, raise the possibility that a shift to non-Pinus feeding triggereda recent bout of ecological speciation as Neodiprion species special-ized on previously inaccessible hosts (i.e., non-Pinus feeding repre-sents a novel adaptive zone sensu Simpson, 1953). Testing thishypothesis will require further clarification of species boundarieswithin the enigmatic sertifer group, increased sampling of loci andpopulations, and methods that can disentangle the effects of lineagesorting, introgression, and phylogenetic history on gene genealogieswithin the Neodiprion species tree (e.g., Hey and Nielsen, 2004; Car-stens and Knowles, 2007; Edwards et al., 2007).

Acknowledgments

We are grateful to the following individuals for providing spec-imens included in this study: B. Fitzgibbon, R. Garbutt, J. Hodge, W.Ingram, S. Li, K. Nystrom, J. Rousselet, G. Sanchez-Martinez, S. Mun-son, L. Pederson, D. Schultz, M. Schultz, G. Turston, D. Whittwer,and E. Wittenmuth. We also thank the following individuals for ad-vice, logistical support, or assistance in the field: C. Asaro, M. Breon,S. Codella, D. Conser, E. Czerwinski, A. Eglitis, W. Ingram, M. Linnen,A. Lynch, B. Mayfield, K. Raffa, D. Smith, L. Thompson, T. Tigner, M.Wagner, and many kind foresters and entomologists. We wouldespecially like to thank D. Smith for his assistance with identifica-tions. In addition, A. Thornton provided lab assistance, and B. Jen-nings provided advice and assistance during the development ofthe anonymous nuclear locus. We also thank two anonymousreviewers for their constructive comments and suggestions. Fund-ing for this research was provided by a Graduate Research Fellow-ship and a Dissertation Improvement Grant (DEB-0308815) fromthe National Science Foundation, a Science to Achieve ResultsGraduate Fellowship from the Environmental Protection Agency,the Putnam Expeditionary Fund at the Museum of ComparativeZoology, the Theodore Roosevelt Memorial Fund at the AmericanMuseum of Natural History, and the Department of Organismicand Evolutionary Biology at Harvard University.

Appendix A

Collection data and GenBank numbers for all specimens

IDA SpeciesB Date Location HostC Accession Nos.D

014-01 N. abbotii* (Leach 1817) 24.vii.01 USA: ME: Mount Desert Island P. resinosa EF361848, EF361983, EF362253,EF362118

016-01 N. abietis (Harris 1841) 25.vii.01 USA: ME: Fort Point State Park; N0 W0 Abies balsamea EU279467, EU279565, EU279663,EU279761

082-02 N. abietis 29.vi.02 Canada: ON: E of Nairn Centre on 17; N46.3334 W81.5722 Abies balsamea EU279496, EU279594, EU279692,EU279790

086-02 N. abietis 29.vi.02 Canada: ON: E of Nairn Centre on 17; N46.3334 W81.5722 Picea glauca EU279499, EU279597, EU279695,EU279793

361-02(a) N. abietis 13.viii.02 Canada: NF: 5km SE of Corner Brook Abies balsamea EU279546, EU279644, EU279742,EU279840

369-02(b) N. abietis 31.vii.02 Canada: NF: Corner Brook Picea glauca EF361962, EF362097, EF362367,EF362232

NAt1A(c) N. abietis 2001 France: St. Pierre et Miquelon (recent introduction) Abies sp. EU279555, EU279653, EU279751,EU279849

NAt2P(c) N. abietis 2001 Canada: ON Picea sp. EU279556, EU279654, EU279752,EU279850

044-03 N. autumnalis Smith andWagner 1986

20.vi.03 USA: AZ: Flagstaff, east side of Spurr Rd. (behind mall); N35.2236W111.5787

P. ponderosa EF361861, EF361996, EF362266,EF362131

060-03 N. autumnalis 25.vi.03 USA: AZ: W of Springerville on 260; N34.0911 W109.4348 P. ponderosa EU279490, EU279588, EU279686,EU279784

061-03 N. autumnalis 26.vi.03 USA: NM: S of Sapello on 518; N35.7503 W105.2503 P. ponderosa EU279492, EU279590, EU279688,EU279786

066-03A N. autumnalis 26.vi.03 USA: NM: S of Sapello on 518; N35.7503 W105.2503 P. ponderosa EU279493, EU279591, EU279689,EU279787

078-03B N. autumnalis 28.vi.03 USA: CO: E of Ault at 90/14; N40.6393 W104.5253 P. ponderosa EU279494, EU279592, EU279690,EU279788

089-04 N. compar* (Leach 1817) 13.vii.04 USA: FL: San Felasco Administrative offices, Gainesville; N29.7476W82.4775

P. palustris EF361877, EF362012, EF362282,EF362147

006-05(c) N. dailingensis Xiao et al. 1985 2005 China: Dandong, Liaoning EF361843, EF361978, EF362248,EF362113

019-02 N. deleoni Ross 1955 15.vi.02 USA: CA: E of Cougar; on NF-42N12; N41.5625 W122.129 Abies concolor EU279470, EU279568, EU279666,EU279764

027-02.2 N. deleoni 15.vi.02 USA: CA: E of Cougar; on NF-42N12; N41.5546 W122.1109 Abies concolor EU279472, EU279570, EU279668,EU279766

121-03.2 N. deleoni 8.vii.03 USA: ID: W of Laird Park on NF-447; N46.9584 W116.6012 Tsugaheterophylla

EU279518, EU279616, EU279714,EU279812

122-03B N. deleoni 8.vii.03 USA: ID: W of Laird Park on NF-447; N46.9584 W116.6012 Abies grandis EU279521, EU279619, EU279717,EU279815

126-03A.1 N. deleoni 10.vii.03 USA: OR: NW of La Grande; NF-31; N45.451 W118.1997 Abies grandis EU279523, EU279621, EU279719,EU279817

126-03A.2 N. deleoni 10.vii.03 USA: OR: NW of La Grande; NF-31; N45.451 W118.1997 Abies grandis EU279524, EU279622, EU279720,EU279818

135-03 N. demoides Ross 1955 11.vii.03 USA: WA: W of Leavenworth, Chatter Creek Campground; N47.606W120.8858

P. monticola EU279535, EU279633, EU279731,EU279829

(continued on next page)

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Appendix A. (continued)

IDA SpeciesB Date Location HostC Accession Nos.D

137-03 N. demoides 11.vii.03 USA: WA: W of Leavenworth, Chatter Creek Campground; N47.6103W120.89

P. contorta EU279538, EU279636, EU279734,EU279832

147-03B N. demoides 14.vii.03 USA: WA: N of Snoquera (McCullough Seed Orchard); N47.0666W121.5872

P. monticola EU279543, EU279641, EU279739,EU279837

162-04 N. dubiosus* Schedl 1933 29.vii.04 USA: ME: near Three Streams on Hardscrabble Rd.; N45.4906W70.236

P. banksiana EF361922, EF362057, EF362327,EF362192

001-03B N. edulicolus Ross 1955 22.iv.03 USA: AZ: S of Twin Arrows on Rd. 233b; N35.0456 W111.3102 P. edulis EU279461, EU279559, EU279657,EU279755

004-03A N. edulicolus 22.iv.03 USA: AZ: E of Sunset Crater on NF-545; N35.4005 W111.4262 P. edulis EU279462, EU279560, EU279658,EU279756

009-03 N. edulicolus 25.iv.03 USA: AZ: N of Jacob Lake on 89A; N36.847 W112.2695 P. edulis EU279463, EU279561, EU279659,EU279757

012-03.1 N. edulicolus 26.iv.03 USA: UT: W of Enterprise on Crestline Rd.; N37.6069 W113.9298 P. monophylla EU279464, EU279562, EU279660,EU279758

012-03.2 N. edulicolus 26.iv.03 USA: UT: W of Enterprise on Crestline Rd.; N37.6069 W113.9298 P. monophylla EU279465, EU279563, EU279661,EU279759

034-04 N. excitans* Rohwer 1921 1.v.04 USA: NC: Richfield, on 49; N35.4772 W80.2553 P. echinata EF361856, EF361991, EF362261,EF362126

023-03 N. gillettei (Rohwer 1908) 29.iv.03 USA: AZ: W of Springerville on 260; N34.0911 W109.4348 P. ponderosa EF361852, EF361987, EF362257,EF362122

010-04 N. hetricki* Ross 1955 26.iv.04 USA: TN: N of Murfreesboro on I-24; N35.9332 W86.5323 P. taeda EF361846, EF361981, EF362251,EF362116

132-04 N. lecontei* (Fitch 1858) 23.vii.04 USA: MD: S of Easton on 50; N38.7162 W76.0639 P. virginiana EF361908, EF362043, EF362313,EF362178

035-0321B(d)

N. maurus* Rohwer 1918 Canada: ON: Kashabowie; N0 W0 P. banksiana EF361858, EF361993, EF362263,EF362128

184-03.1 N. merkeli merkeli* Ross 1961 26.xi.03 USA: FL: Palmdale, on 74; N26.9228 W81.3366 P. elliottii EF361935, EF362070, EF362340,EF362205

017-02 N. mundus Rohwer 1918 15.vi.02 USA: CA: NE of Weed on 97; N41.5446 W122.2321 P. ponderosa EU279469, EU279567, EU279665,EU279763

110-03 N. mundus 7.vii.03 USA: ID: Coeur D’Alene, Kathleen Ave.; N47.7171 W116.8149 P. contorta EU279509, EU279607, EU279705,EU279803

129-03A N. mundus 10.vii.03 USA: OR: NW of La Grande; NF-31; N45.439 W118.2282 P. ponderosa EU279526, EU279624, EU279722,EU279820

132-03 N. mundus 10.vii.03 USA: OR: W of La Grande, at NF-21/NF-450; N45.3374 W118.3177 P. contorta EU279532, EU279630, EU279728,EU279826

038-02 N. nanulus contortae Ross1955

16.vi.02 USA: CA: W of Dunsmuir on Castle Lake Rd.; N41.2384 W122.3814 P. contorta EU279476, EU279574, EU279672,EU279770

009-04 N. nanulus nanulus Schedl1933

26.iv.04 USA: TN: Buffalo Valley; I-40W rest stop; N36.1397 W85.8064 P. virginiana EF361844, EF361979, EF362249,EF362114

026-04 N. nanulus nanulus 30.iv.04 USA: NC: NW of Longtown on Old Hwy 105; N35.8011 W81.9338 P. pungens EU279471, EU279569, EU279667,EU279765

060-04 N. nanulus nanulus 4.v.04 USA: VA: Dinwiddie on 226; N37.2094 W77.4744 P. taeda EU279491, EU279589, EU279687,EU279785

080-02 N. nanulus nanulus 29.vi.02 Canada: ON: W of Nairn Centre on 17; N46.3113 W81.6553 P. banksiana EU279495, EU279593, EU279691,EU279789

088-02 N. nanulus nanulus 29.vi.02 Canada: ON: SE of Cartier at 144/Old Cartier Rd.; N46.6244 W81.454 P. banksiana EF361876, EF362011, EF362281,EF362146

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olecularPhylogenetics

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154-04 N. nanulus nanulus 28.vii.04 USA: ME: Great Wass Island (Nature Conservancy trail); N44.481W67.5946

P. banksiana EF361916, EF362051, EF362321,EF362186

209-04 N. nigroscutum* Middleton1933

17.viii.04 USA: WI: N of Butternut on 13; N46.0865 W90.5507 P. banksiana EF361945, EF362080, EF362350,EF362215

040-03 N. nr. abietis 1 18.vi.03 USA: AZ: N of Grand Canyon North Rim, near Dog Lake trail head;N36.4172 W112.0915

Picea sp. EU279478, EU279576, EU279674,EU279772

042-03 N. nr. abietis 1 18.vi.03 USA: AZ: N of Grand Canyon North Rim, near Dog Lake trail head;N36.4172 W112.0915

Picea sp. EU279479, EU279577, EU279675,EU279773

055-03 N. nr. abietis 1 22.vi.03 USA: AZ: SW of Safford on 366 (Pinaleno Mtns); N32.7158W109.9397

Picea engelmani EU279487, EU279585, EU279683,EU279781

059-03 N. nr. abietis 1 24.vi.03 USA: AZ: NE of Payson off NF-300; N34.3684 W111.0111 Abies concolor EU279489, EU279587, EU279685,EU279783

027-02.3 N. nr. abietis 2 15.vi.02 USA: CA: E of Cougar; on NF-42N12; N41.5546 W122.1109 Abies concolor EU279473, EU279571, EU279669,EU279767

122-03A N. nr. abietis 2 8.vii.03 USA: ID: W of Laird Park on NF-447; N46.9584 W116.6012 Abies grandis EU279520, EU279618, EU279716,EU279814

126-03B N. nr. abietis 2 10.vii.03 USA: OR: NW of La Grande; NF-31; N45.451 W118.1997 Abies grandis EU279525, EU279623, EU279721,EU279819

084-03A N. nr. demoides 30.vi.03 USA: WY: N of Buford at 210/Vedauwoo Rd.; N41.191 W105.2956 P. flexilis EU279498, EU279596, EU279694,EU279792

090-03 N. nr. demoides 1.vii.03 USA: WY: N of Buford at 210/Vedauwoo Rd.; N41.191 W105.2956 P. flexilis EU279501, EU279599, EU279697,EU279795

033-03 N. nr. edulicolus 2.v.03 USA: AZ: NE of Douglas on Price Canyon Rd.; N31.7476 W109.3533 P. leiophylla EU279474, EU279572, EU279670,EU279768

048-03A N. nr. mundus 20.vi.03 USA: AZ: near Maine on Pineaire Access Rd.; N35.233 W111.9702 P. ponderosa EU279482, EU279580, EU279678,EU279776

068-04(e) N. nr. omosus 1.ii.04 Mexico: Aguascalientes: Sierra Fria P. michoacana EF361870, EF362275, EF362140,EF362005

113-04 N. pinetum* (Norton 1869) 19.vii.04 USA: TN: Crossville, on 298 S of I-40; N35.98 W85.0152 P. strobus EF361895, EF362030, EF362300,EF362165

148-04 N. pinusrigidae* (Norton 1868) 27.vii.04 USA: ME: Cornish, on 25; N43.8052 W70.8166 P. rigida EF361914, EF362049, EF362319,EF362184

043-04.2 N. pratti pratti* (Dyar 1899) 3.v.04 USA: NC: Roanoke Rapids on 158; N36.4379 W77.6533 P. virginiana EF361860, EF361995, EF362265,EF362130

183-02 N. rugifrons* Middleton 1933 10.viii.02 Canada: ON: near Britt on 69; N45.7987 W80.5352 P. banksiana EF361933, EF362068, EF362338,EF362203

040-02 N. scutellatus Rohwer 1918 17.vi.02 USA: OR: W of Fort Klamath on NF-3384; N42.7041 W122.0879 Pseudotsugamenziesii

EU279477, EU279575, EU279673,EU279771

052-02B N. scutellatus 20.vi.02 USA: WA: SE of Greenwater near 410/NF-70; N47.1386 W121.6241 Pseudotsugamenziesii

EU279485, EU279583, EU279681,EU279779

136-03B N. scutellatus 11.vii.03 USA: WA: W of Leavenworth, Chatter Creek Campground; N47.6103W120.89

Pseudotsugamenziesii

EU279537, EU279635, EU279733,EU279831

141-03D N. scutellatus 12.vii.03 USA: WA: SE of Van Zandt; N48.7697 W122.1664 Pseudotsugamenziesii

EU279541, EU279639, EU279737,EU279835

142-03B N. scutellatus 12.vii.03 USA: WA: SE of Van Zandt; N48.7697 W122.1664 Pseudotsugamenziesii

EU279542, EU279640, EU279738,EU279836

143-03 N. scutellatus 12.vii.03 USA: WA: SE of Van Zandt; N48.7697 W122.1664 Pseudotsugamenziesii

EF361911, EF362046, EF362316,EF362181

069-02 N. sertifer (Geoffroy 1785) 26.vi.02 Canada: ON: E of Bancroft on 28; N45.0659 W77.7491 P. mugho EF361871, EF362006, EF362276,EF362141

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Appendix A. (continued)

IDA SpeciesB Date Location HostC Accession Nos.D

122-02(f) N. sertifer 24.vi.02 Canada: ON: NW of Ingleside, St. Georges Cemetery; N45.0549W75.0857

P. sylvestris EU279519, EU279617, EU279715,EU279813

NSt3(c) N. sertifer 2001 France Pinus sp. EU279557, EU279655, EU279753,EU279851

NSt4(c) N. sertifer 2001 France Pinus sp. EU279558, EU279656, EU279754,EU279852

014-03B N. sp. 27.iv.03 USA: NM: S of Sapello on 518; N35.7503 W105.2503 P. ponderosa EU279466, EU279564, EU279662,EU279760

043-02 N. sp. 18.vi.02 USA: OR: W of Camp Sherman on NF-1210; N44.451 W121.7248 Abies concolor EU279480, EU279578, EU279676,EU279774

048-03B N. sp. 20.vi.03 USA: AZ: near Maine on Pineaire Access Rd.; N35.233 W111.9702 P. ponderosa EU279483, EU279581, EU279679,EU279777

052-02A N. sp. 20.vi.02 USA: WA: SE of Greenwater near 410/NF-70; N47.1386 W121.6241 Pseudotsugamenziesii

EU279484, EU279582, EU279680,EU279778

054-03 N. sp. 22.vi.03 USA: AZ: SW of Safford on 366 (Pinaleno Mtns); N32.7158W109.9397

Pseudotsugamenziesii

EU279486, EU279584, EU279682,EU279780

082-03 N. sp. 28.vi.03 USA: CO: S of Indian Meadows on 63E; N40.6088 W105.5349 P. contorta EU279497, EU279595, EU279693,EU279791

086-03 N. sp. 1.vii.03 USA: WY: N of Buford at 210/Vedauwoo Rd.; N41.191 W105.2956 P. flexilis EU279500, EU279598, EU279696,EU279794

091-03A N. sp. 2.vii.03 USA: WY: just W of Lusk on 18/20; N42.7506 W104.4667 P. ponderosa EU279502, EU279600, EU279698,EU279796

093-03 N. sp. 2.vii.03 USA: WY: just W of Lusk on 18/20; N42.7506 W104.4667 P. ponderosa EU279503, EU279601, EU279699,EU279797

098-03 N. sp. 3.vii.03 USA: MT: W of Bonner at Deer Creek underpass; N46.8774W113.9132

P. ponderosa EU279504, EU279602, EU279700,EU279798

101-03 N. sp. 4.vii.03 USA: MT: E of Lincoln on 200; N46.9517 W112.7345 P. contorta EU279505, EU279603, EU279701,EU279799

102-03 N. sp. 4.vii.03 USA: MT: E of Lincoln on 200; N46.9517 W112.7345 P. contorta EU279506, EU279604, EU279702,EU279800

104-03 N. sp. 5.vii.03 USA: MT: W of Paradise on 200; N47.5829 W115.2889 P. ponderosa EU279507, EU279605, EU279703,EU279801

109-03 N. sp. 6.vii.03 USA: WA: S of Deer Park on 395; N47.9109 W117.4457 P. ponderosa EU279508, EU279606, EU279704,EU279802

111-03 N. sp. 7.vii.03 USA: ID: Coeur D’Alene at 95/53; N47.823 W116.7889 P. ponderosa EU279510, EU279608, EU279706,EU279804

116-03 N. sp. 7.vii.03 USA: ID: N of Hayden on 95; N47.7826 W116.7893 P. ponderosa EU279511, EU279609, EU279707,EU279805

117-03.1 N. sp. 7.vii.03 USA: ID: N of Hayden on 95; N47.7826 W116.7893 P. ponderosa EU279512, EU279610, EU279708,EU279806

117-03.2 N. sp. 7.vii.03 USA: ID: N of Hayden on 95; N47.7826 W116.7893 P. ponderosa EU279513, EU279611, EU279709,EU279807

119-03A.1 N. sp. 8.vii.03 USA: ID: S of Coeur D’Alene on 95; N47.6361 W116.8648 P. ponderosa EU279514, EU279612, EU279710,EU279808

119-03A.3 N. sp. 8.vii.03 USA: ID: S of Coeur D’Alene on 95; N47.6361 W116.8648 P. ponderosa EU279515, EU279613, EU279711,EU279809

119-03B N. sp. 8.vii.03 USA: ID: S of Coeur D’Alene on 95; N47.6361 W116.8648 P. ponderosa EU279516, EU279614, EU279712,EU279810

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130-03 N. sp. 10.vii.03 USA: OR: NW of La Grande; NF-31; N45.439 W118.2282 P. ponderosa EU279527, EU279625, EU279723,EU279821

131-03 N. sp. 10.vii.03 USA: OR: W of La Grande, at NF-21/NF-450; N45.3374 W118.3177 P. contorta EU279531, EU279629, EU279727,EU279825

134-03A N. sp. 11.vii.03 USA: WA: W of Leavenworth, Chatter Creek Campground; N47.606W120.8858

Tsugaheterophylla

EU279533, EU279631, EU279729,EU279827

136-03A N. sp. 11.vii.03 USA: WA: W of Leavenworth, Chatter Creek Campground; N47.6103W120.89

Pseudotsugamenziesii

EU279536, EU279634, EU279732,EU279830

138-03 N. sp. 11.vii.03 USA: WA: W of Leavenworth, Chatter Creek Campground; N47.6103W120.89

P. contorta EU279539, EU279637, EU279735,EU279833

141-03A N. sp. 12.vii.03 USA: WA: SE of Van Zandt; N48.7697 W122.1664 Pseudotsugamenziesii

EU279540, EU279638, EU279736,EU279834

148-03 N. sp. 14.vii.03 USA: WA: N of Snoquera (McCullough Seed Orchard); N47.0666W121.5872

P. monticola EU279544, EU279642, EU279740,EU279838

149-03(g) N. sp. 16.vii.03 USA: CA: near Selad Valley P. attenuata EU279545, EU279643, EU279741,EU279839

002-04B N. species 1* 25.iv.04 USA: AR: NW of Artesian on CR-193; N33.4119 W92.4915 P. taeda EF361839, EF361974, EF362244,EF362109

081-04 N. species 2* 11.vii.04 USA: FL: N of Altoona on SR 19; N29.0339 W81.6399 P. clausa EF361874, EF362009, EF362279,EF362144

180-04 N. swainei* Middleton 1931 14.viii.04 USA: WI: E of Mondovi on 10; N44.5748 W91.6023 P. banksiana EF361932, EF362067, EF362337,EF362202

002-04A N. taedae linearis* Ross 1955 25.iv.04 USA: AR: NW of Artesian on CR-193; N33.4119 W92.4915 P. taeda EF361838, EF361973, EF362243,EF362108

121-03.1 N. tsugae Middleton 1933 8.vii.03 USA: ID: W of Laird Park on NF-447; N46.9584 W116.6012 Tsugaheterophylla

EU279517, EU279615, EU279713,EU279811

131-02(h) N. tsugae 18.vii.02 USA: AK: Cholmondeley Sound, Prince of Wales Island — EU279528, EU279626, EU279724,EU279822

131-02.2(h) N. tsugae 18.vii.02 USA: AK: Cholmondeley Sound, Prince of Wales Island — EU279529, EU279627, EU279725,EU279823

131-02.3(h) N. tsugae 18.vii.02 USA: AK: Cholmondeley Sound, Prince of Wales Island — EU279530, EU279628, EU279726,EU279824

134-03B N. tsugae 11.vii.03 USA: WA: W of Leavenworth, Chatter Creek Campground; N47.606W120.8858

Tsugaheterophylla

EU279534, EU279632, EU279730,EU279828

500-02-030(i)

N. tsugae 31.vii.02 Canada: BC: Keen Creek (‘‘Plot#030”); N49.9022 W116.934 Tsugaheterophylla

EU279547, EU279645, EU279743,EU279841

500-02-030.2(i)

N. tsugae 31.vii.02 Canada: BC: Keen Creek (‘‘Plot#030”); N49.9022 W116.934 Tsugaheterophylla

EU279548, EU279646, EU279744,EU279842

500-02-074.1(i)

N. tsugae 30.vii.02 Canada: BC: Goldstream R. (‘‘Plot#074”); N51.6047 W118.592 Tsugaheterophylla

EU279549, EU279647, EU279745,EU279843

500-02-074.4(i)

N. tsugae 30.vii.02 Canada: BC: Goldstream R. (‘‘Plot#074”); N51.6047 W118.592 Tsugaheterophylla

EU279550, EU279648, EU279746,EU279844

500-02-086.1(i)

N. tsugae 29.vii.02 Canada: BC: Glacier National Park (‘‘Plot#086”); N51.4868W117.4722

Tsugaheterophylla

EU279551, EU279649, EU279747,EU279845

500-02-086.3(i)

N. tsugae 29.vii.02 Canada: BC: Glacier National Park (‘‘Plot#086”); N51.4868W117.4722

Tsugaheterophylla

EU279552, EU279650, EU279748,EU279846

504-02(j) N. tsugae 6.viii.02 Canada: BC: Km 2; Phantom Main QCI; Queen Charlotte City (P.O.) Tsugaheterophylla

EU279553, EU279651, EU279749,EU279847

(continued on next page)

C.R.Linnen,B.D

.Farrell/Molecular

Phylogeneticsand

Evolution48

(2008)240–

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256 C.R. Linnen, B.D. Farrell / Molecular Phylogenetics and Evolution 48 (2008) 240–257

Appendix B. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2008.03.021.

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