BMC Evolutionary Biology BioMed Central
BMC Evolutionary BiologyBioMed CentralOpen AccessResearch articleImpact of duplicate gene copies on phylogenetic analysis anddivergence time estimates in butterfliesNélida Pohl, Marilou P Sison-Mangus, Emily N Yee, Saif W Liswi andAdriana D Briscoe*Address: Department of Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA 92697, USAEmail: Nélida Pohl - npohl@uci.edu; Marilou P Sison-Mangus - msisonma@uci.edu; Emily N Yee - enyee@uci.edu;Saif W Liswi - sliswi@gmail.com; Adriana D Briscoe* - abriscoe@uci.edu* Corresponding authorPublished: 13 May 2009BMC Evolutionary Biology 2009, 9:99doi:10.1186/1471-2148-9-99Received: 10 November 2008Accepted: 13 May 2009This article is available from: http://www.biomedcentral.com/1471-2148/9/99 2009 Pohl et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractBackground: The increase in availability of genomic sequences for a wide range of organisms hasrevealed gene duplication to be a relatively common event. Encounters with duplicate gene copieshave consequently become almost inevitable in the context of collecting gene sequences forinferring species trees. Here we examine the effect of incorporating duplicate gene copies evolvingat different rates on tree reconstruction and time estimation of recent and deep divergences inbutterflies.Results: Sequences from ultraviolet-sensitive (UVRh), blue-sensitive (BRh), and long-wavelengthsensitive (LWRh) opsins, EF-1 and COI were obtained from 27 taxa representing the five majorbutterfly families (5535 bp total). Both BRh and LWRh are present in multiple copies in somebutterfly lineages and the different copies evolve at different rates. Regardless of the phylogeneticreconstruction method used, we found that analyses of combined data sets using either slower orfaster evolving copies of duplicate genes resulted in a single topology in agreement with our currentunderstanding of butterfly family relationships based on morphology and molecules. Interestingly,individual analyses of BRh and LWRh sequences also recovered these family-level relationships. Twodifferent relaxed clock methods resulted in similar divergence time estimates at the shallowernodes in the tree, regardless of whether faster or slower evolving copies were used, with largerdiscrepancies observed at deeper nodes in the phylogeny. The time of divergence between themonarch butterfly Danaus plexippus and the queen D. gilippus (15.3–35.6 Mya) was found to be mucholder than the time of divergence between monarch co-mimic Limenitis archippus and red-spottedpurple L. arthemis (4.7–13.6 Mya), and overlapping with the time of divergence of the co-mimeticpassionflower butterflies Heliconius erato and H. melpomene (13.5–26.1 Mya). Our family-levelresults are congruent with recent estimates found in the literature and indicate an age of 84–113million years for the divergence of all butterfly families.Conclusion: These results are consistent with diversification of the butterfly families following theradiation of angiosperms and suggest that some classes of opsin genes may be usefully employedfor both phylogenetic reconstruction and divergence time estimation.Page 1 of 16(page number not for citation purposes)
BMC Evolutionary Biology 2009, 9:99BackgroundGene duplication has long been recognized as a majorsource of evolutionary innovation [1]. It is a pervasiveevolutionary process, with 50% of all genes in any givengenome expected to duplicate and proliferate at least oncein time scales ranging from 35 to 350 MA [2]. In molecular phylogenetics, gene duplication is a process that canlead to discordance between gene and species trees, andprevious work has shown large problems with duplicategenes undergoing concerted evolution or birth-and-deathprocesses [3]. The consensus has been to avoid the use ofparalogous genes until methods are developed to handletheir potential confounding effects [4]. Given the highlikelihood of gene duplication under neutral evolutionaryprocesses, however, as the size of molecular data sets getslarger (in number of genes and taxa used) the amplification of duplicated genes may happen inadvertently evenin the course of targeting genes that appear at first glanceto be single copy (See below). The challenge is to beginstudying the range of phylogenetic signal such duplicategenes provide, to assess their evolutionary dynamics andpotential signal for phylogenetics. Within the butterflies,for instance, duplicate copies of opsin genes have beenfound both within and between families [5], and our previous work suggested the possible utility of one of theopsins for phylogenetic purposes [6]. The current workseeks to clarify the potential utility of an expanded collection of opsins for butterfly phylogenetic reconstructionand divergence time estimation.Historically opsin genes have been advocated as phylogenetic markers, due to the amount of information we possess about their molecular evolution relative to othernuclear genes, and the wealth of cloned sequences available for a wide array of organisms [7]. In fact, the longwavelength-sensitive opsin gene (LWRh) has routinelybeen used for the past 10 years in bee, bumblebee andwasp phylogenetic studies [8-12], and has proven usefulat both shallow [13] and deep phylogenetic levels, suggesting its utility at resolving family level, Cretaceous ageinsect divergences [14,15]. After gaining momentum as aphylogenetic marker, a second copy of the gene, LWRh2,was subsequently discovered in bees, which fortunatelyappears to have evolved under a trajectory independent ofthe first copy, making both copies suitable for tree building [16]. Currently there is little information about thepotential use of other opsin genes in reconstructing insectphylogenies [16], yet almost all insects that have beenstudied including butterflies have three clades of opsinsthat encode spectrally distinct visual pigments present inthe adult compound eye that are ultraviolet- (UVRh),blue- (BRh) and long wavelength (LWRh)-sensitive [17].This suggests that other clades of opsins may also be useful for phylogenetic reconstruction over a similar range ofdivergence utterflies are some of the best known organisms, possessing remarkable life histories and an uncanny beauty, butyet their most basal relationships have been until recentlystill obscure [18]. In the early days of butterfly evolutionresearch, the study of the oldest butterfly lineages wasintertwined with speculations about timing of their origins [19-22]. In more recent studies, the complexity ofsimply finding the most plausible topologies, and the difficulty of disentangling molecular evolutionary rates anddivergence times, resulted in few studies directly concerned with timing the divergence of butterfly clades.Concerns about the applicability of a molecular clock[23], and the scarcity of butterfly fossils with which to calibrate it [24-26] have also undoubtedly contributed tothis paucity in the literature. In recent years the advent ofboth non-parametric and highly parametric Bayesian[27,28] methods that free the estimations of divergencetimes from the restrictions of a molecular clock and permit the incorporation of flexible fossil or biogeographicalcalibration points, has rekindled efforts to date the different diversification events within butterflies, and in theprocess sparked a controversy about when and where butterflies originated [27,29]. A generally young and scantrecord comprised of about 50 Rhopaloceran fossils, agroup which includes the skippers (Hesperioidea), nocturnal butterflies (Hedyloidea) and true dayflying butterflies (Papilionoidea), all found within the Cenozoic Era(65.5-0 Mya) and not older than 56–57 Ma (a skipper)and 48 Ma (a papilionid), respectively [30,31], has in ofitself not been particularly useful in the direct estimationof the earliest butterfly divergences; and is considered bysome researchers as a veritable indicator of a recent butterfly origin, dating back to the last epoch of the late Cretaceous (70.6 0.6 – 65.8 0.3 Mya) or early Cenozoic(65.5 – 0.0 Mya) no earlier than 70 Ma ago [18,29]. Onthe other hand, molecular phylogenetic methods haveproduced much older divergence time estimates for several butterfly families, prompting many to ascribe the origin of butterflies to the diversification of angiosperms,between 100 and 140 Mya [32-37].Another group of insects thought to have evolved concordantly with the early diversification of angiosperms arethe ants [38], but despite thorough sampling and anample fossil record, disagreements concerning basal relationships and timing of the earliest divergences still exist[39,40](but see [41]). In contrast, the basal relationshipsof butterflies are for the most part resolved, with our current understanding of relationships at the familial levelbeing based on the study of Wahlberg and collaborators,which employed both molecular and morphological datato resolve deep nodes in the phylogeny of butterflies [42].Therefore, with a known phylogeny, butterflies are a useful group of organisms for examining the impact of dupli-Page 2 of 16(page number not for citation purposes)
BMC Evolutionary Biology 2009, 9:99cate genes on phylogenetic reconstruction and divergencetime estimation.In this study, we examine the effect of including duplicated opsin genes evolving at different rates on phylogenetic reconstruction and divergence time estimates. Wefind that individual and combined analyses of the BRhand LWRh genes are able to recover butterfly family-levelrelationships where previously morphological characterswere required in addition to molecular [42]. We estimatedivergence times for clades of high interest to the ecologyand evolutionary biology communities, such as for the comimics Heliconius erato and H. melpomene [43], the migratory monarch Danaus plexippus and the non-migratoryqueen D. gilippus [44] and their co-mimics in the genusLimenitis [45-47]. We find our estimates of family-leveltimes of divergence with slower evolving gene duplicatesto mostly be in agreement with other recent estimatesfound in the literature based on molecular data, and wepush back the minimum age of divergence for the mostbasal butterfly family to 113 Mya. Our results suggest thepotential utility of the opsins for resolving even older andmore complex group relationships such as the moths.Results and discussionRelative rates tests classify duplicate BRh and LWRhopsins functionallyAs mentioned previously, most insects including butterflies have adult compound eyes that contain at least threeclasses of opsin genes (UVRh, BRh and LWRh) that encodevisual pigments with wavelengths of peak absorbance, max, that fall roughly into the UV (300–400 nm), blue(400–500 nm) and long wavelength (500–600 nm) portions of the light spectrum. Within these broad partitionsof the spectrum, the visual pigments of butterflies typically cluster in narrower ranges (i.e., 345–380 nm, 437–470 nm and 514–565) (reviewed in [48]). There are, however, additional opsins that evolved from these three basicclasses in some butterfly eyes, which encode visual pigments with max values outside of these typical ranges (seebelow).All three basic classes of opsin (UVRh, BRh and LWRh)were present in all 27 butterfly species included in thisstudy [Additional File 1], except in the two closely-relatedsatyrines, Neominois ridingsii and Oeneis chryxus, in whichno BRh gene could be found after exhaustive screening ofhead-specific cDNAs. We think that these species probably do have blue-sensitive visual pigments in the eyebased on physiological studies (Gary Bernard, pers.comm.) but we were simply unsuccessful in retrievingthem. Similarly, we also think that besides the violet opsinwe found in the pierid Colias philodice this species likelyhas a blue-sensitive visual pigment in the eye based onelectrophysiological studies of a related species [49] buthttp://www.biomedcentral.com/1471-2148/9/99we did not find it. Full-length coding regions were otherwise obtained for the opsin cDNAs, including both startand stop codons. The size of these transcripts, including 3'and 5' UTR regions ranged from 1137–1575 bp (UVRh),996–1590 bp (BRh), and 1143–1743 bp (LWRh). Besidesthe three basic opsin classes, all lycaenid butterflies (thisstudy and [50]) and the pierid Pieris rapae [51] have duplicated blue opsins, representing two independent BRhduplications, while the papilionids Papilio xuthus and P.glaucus, the riodinid Apodemia mormo and the moth Bombyx mori possess duplicated LWRh opsins [6,52,53] representing four independent gene duplications (gene namesfor the long-wavelength pigments have been renamedhere for simplicity).Relative rates test showed that of the lycaenid blue opsinduplicates, BRh1 evolved slower than BRh2 in every one ofthe 7 lycaenid species sampled, although not significantlyso (Table 1). The slower rate of evolution of the BRh1opsin is consistent with our observation that this geneencodes the 437 nm visual pigment in Lycaena rubidus,which is a wavelength of peak absorbance that is moretypical of the "blue-sensitive" visual pigments in butterflies, than the duplicate BRh2 gene in L. rubidus whichencodes the unusually red-shifted 500 nm visual pigment[50]. Similarly, in the pierid Pieris rapae, the blue opsincopy (B), which encodes a 450 nm visual pigment [51]evolved slower than the violet copy (V), which encodes amore unusual 425 nm pigment, but this result was not significant either. Among the duplicated LWRh genes, significantly different rates of evolution were observed whereBombyx mori LWRh2 evolved slower than B. mori LWRh1,Papilio LWRh2 evolved slower than both Papilio LWRh1and Papilio LWRh3, and Apodemia mormo LWRh1 evolvedslower than A. mormo LWRh2 (Table 1). Here too, theslowest evolving Papilio LWRh2 encodes a pigment thatwith a max at 520 nm is functionally more similar to otherbutterfly long-wavelength sensitive visual pigments in itswavelength of peak absorbance than the pigment encodedby Papilio LWRh3 ( max 575 nm), similarly, the slowerevolving Apodemia LWRh1 encodes a pigment that with aslightly blue-shifted max at 505 nm is much more typicalof other butterfly pigments than its faster evolving LWRh2copy that encodes a pigment with the highly atypical maxat 600 nm [6,54]. Given that the relative rates tests seemable to classify the slowest and fastest evolving gene copies in a way that also roughly reflected their function, withthe slowest evolving copies having spectral properties falling in a more narrow, similar and presumably ancestralrange than the fastest evolving copies, we decided todivide our data for further analysis (see below) into alignments which included the slowest or fastest evolving copies.Page 3 of 16(page number not for citation purposes)
BMC Evolutionary Biology 2009, le 1: Tajima relative rates tests between duplicated copies of BRh and LWRh genes.SpeciesL. rubidusL. heteroneaL. helloidesL. nivalisP. icarusA. glandonS. behriiP. rapaeGene copies (seq A/B)Outgroup (seq C)N sitesUnique differences seq AUnique differences seq BUnique differences seq C 2 (1 d.f.)PBRh1/2A. mormo11271011061220.120.728BRh1/2A. mormo1126981191252.030.154BRh1/2A. mormo994801031092.890.089BRh1/2A. mormo1129901151233.050.081BRh1/2A. mormo11151121401103.110.078BRh1/2A. mormo11171131381122.490.115BRh1/2A. mormo1139971131351.220.270BRhV/BP. xuthus11251251201150.100.750B. moriA. mormoP. xuthusP. xuthusP. xuthusP. glaucusP. glaucusP. glaucusLWRh1/2M. sexta1128107671149.200.002LWRh1/2D. plexippus1109971351236.220.0126LWRh1/2P. rapae11371458811613.940.0002LWRh1/3P. rapae11371141031410.560.455LWRh2/3P. rapae1137971431058.820.003LWRh1/2P. rapae11371568810418.950.00001LWRh1/3P. rapae11371171031450.890.345LWRh2/3P. rapae11379014410612.460.0004SpeciesGene copies (seq A/B)Outgroup (seq C)N sitesUnique differences seq AUnique differences seq BUnique differences seq C 2 (1 d.f.)PP-values of gene pairs evolving at different rates are shown in bold.For phylogenetic analysis and divergence time estimation,we also obtained EF-1 and COI for individual taxawhere such sequences were not already available in GenBank. A total of 66 new gene sequences, including 38opsin genes, 15 EF-1 and 13 COI sequences, are reportedin this study (See Additional File 2). Accession numbersfor all new sequences and those downloaded from GenBank are shown in Additional File 1. The combined dataset consisted of a total of 5523 bp, of which 1158, 1156,1164, 1066 and 982 bp, belonged to the UVRh, BRh,LWRh, EF-1 and COI genes respectively.Maximum parsimony analysis recovers a single tree forbutterfliesMaximum parsimony (MP) analysis of all five genes usingthe slowest evolving opsin duplicates identified by the relative rates test resulted in a single tree (Figure 1, Additional File 1) with a topology congruent with that inferredin a previous study from molecular and morphologicaldata [42]. Re-running this analysis using the faster evolving gene copies also revealed the same topology. As traditionally recognized, Papilionidae is placed as sister to(Pieridae (Nymphalidae (Lycaenidae Riodinidae))).The relationships recovered within Nymphalidae also correspond to the current consensus, which groups Limenitidinae with Heliconiinae and Nymphalinae as sister tothe previous two, Satyrinae as sister to (Nymphalinae (Limenitidinae Heliconiinae)) and Danainae as thebasal subfamily, sister to (Satyrinae (Nymphalinae (Limenitidinae Heliconiinae))). Within Lycaenidae, theTheclinae clade, represented by the hairstreak Satyriumbehrii, groups together with the Lycaeninae, and these twoclades are sister to the Polyommatinae.Dissecting the contribution of different genes to this topological hypothesis through partitioned analysis revealsthat the LWRh gene provides the strongest support to thetopology, followed by the BRh, UVRh, EF-1 and COIgenes (Table 2). This holds true for both combined analyses which included slower and faster gene copies of duplicate genes. Using the slower evolving copies we can seethat both LWRh and BRh support all nodes, as shown bypositive partitioned Bremer support values, whereas theinformation provided by UVRh, EF-1 and COI conflictswith one or more nodes. The UV opsin data stronglyrenders the grouping of both (Lycaenidae Riodinidae),and (Nymphalidae (Lycaenidae Riodinidae)) spurious, and also does not support the monophyly of Satyrinae. EF-1 does not recover the (Lycaeninae Theclinae)clade, but it does not take many steps to retrieve it (partitioned Bremer support -2, Table 2). COI conflicts with 10out of the 26 total nodes, resulting in many negative support values (Table 2). This is not surprising since it haslong been recognized that its fast rate of evolution rendersCOI of limited use when reconstructing phylogenetic history at levels deeper than species [33,55].The Bremer support values rendered by the analysis of thecombined data set using faster evolving gene copies arequalitatively and quantitatively similar to the values of theslower evolving copies in most cases (Table 2). A fewexceptions occur at basal nodes in the tree, where usingPage 4 of 16(page number not for citation purposes)
BMC Evolutionary Biology 2009, ues for these gene partitions approach zero in bothslow and fast gene copy analyses of these nodes.Nymphalinae 60E uphydryas chalcedona20V anessa carduiE . chalcedona21198421234L. arthemisNymphalis antiopaLimenitis arthemis17Limenitis archippus43Limenitidinae18S peyeria mormonia4316Heliconiinae 152215Agraulis vanillae1572S
BMC Evolutionary Biology Research article Open Access Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies Nélida Pohl, Marilou P Sison-Mangus, Emily N Yee, Saif W Liswi and Adriana D Briscoe* Address: Department of Ecology and Evolutionary Biology,
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