© 2004 Society of Systematic Biologists
Phylogeny of Agrodiaetus Hübner 1822 (Lepidoptera: Lycaenidae) Inferred from mtDNA Sequences of COI and COII and Nuclear Sequences of EF1-
: Karyotype Diversification and Species Radiation
Edited by
Ted Schultz: Associate Editor
1 Department of Organismal and Evolutionary Biology, Harvard University 26 Oxford Street, Cambridge, Massachusetts 02138, USA; E-mail: kandul{at}fas.harvard.edu (N.P.K.)
2 Department of Entomology, St. Petersburg State University Universitetskaya nab. 7/9, St. Petersburg 199034, Russia
3 Center for Conservation Biology, Department of Biological Sciences, Stanford University Palo Alto California 94305, USA
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionAppendix 1AcknowledgmentReferences |
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Butterflies in the large Palearctic genus Agrodiaetus (Lepidoptera: Lycaenidae) are extremely uniform and exhibit few distinguishing morphological characters. However, these insects are distinctive in one respect: as a group they possess among the greatest interspecific karyotype diversity in the animal kingdom, with chromosome numbers (n) ranging from 10 to 125. The monophyly of Agrodiaetus and its systematic position relative to other groups within the section Polyommatus have been controversial. Characters from the mitochondrial genes for cytochrome oxidases I and II and from the nuclear gene for elongation factor 1
were used to reconstruct the phylogeny of Agrodiaetus using maximum parsimony and Bayesian phylogenetic methods. Ninety-one individuals, encompassing most of the taxonomic diversity of Agrodiaetus, and representatives of 14 related genera were included in this analysis. Our data indicate that Agrodiaetus is monophyletic. Representatives of the genus Polyommatus (sensu stricto) are the closest relatives. The sequences of the Agrodiaetus taxa in this analysis are tentatively arranged into 12 clades, only 1 of which corresponds to a species group traditionally recognized in Agrodiaetus. Heterogeneous substitution rates across a recovered topology were homogenized with a nonparametric rate-smoothing algorithm before the application of a molecular clock. Two published estimates of substitution rates dated the origin of Agrodiaetus between 2.51 and 3.85 million years ago. During this time, there was heterogeneity in the rate and direction of karyotype evolution among lineages within the genus. Karyotype instability has evolved independently three times in the section Polyommatus, within the lineages Agrodiaetus, Lysandra, and Plebicula. Rapid karyotype diversification may have played a significant role in the radiation of the genus Agrodiaetus.
Keywords: Agrodiaetus; cytochrome oxidase I; cytochrome oxidase II; elongation factor 1; karyotype diversification; phylogeny; Polyommatus; speciation
Received April 1, 2003; Revised August 13, 2003; Accepted November 17, 2003
According to the biological species concept, "species are groups of interbreeding natural populations that are reproductively isolated from other such groups" (Mayr, 1970:12). In other words, species are closed genetic systems with unique gene pools. The appearance of new species is accompanied by the establishment of reproductive barriers that are typically viewed as the consequence of geographic isolation of nascent species (Mayr, 1970; White, 1973). Hybrids between species with different karyotypes may have reduced fertility due to problems in meiotic segregation (White, 1973; Gropp et al., 1982; John et al., 1983; King, 1993). Thus, fixed chromosome differences between species may contribute to postzygotic barriers to gene flow. The role of chromosome rearrangements in maintaining postzygotic isolation between well-established species is not controversial, but whether such rearrangements also play a causal role in the initial stages of speciation has excited vigorous scientific debate. The spectrum of views on this problem is broad, from the general denial of their causal role in animals, accepting only polyploidy and special cases such as monobrachial centric fusion (Coyne and Orr, 1998; Spirito, 1998) to the claim that isolation by chromosomal rearrangements could be a major mode of speciation (White, 1973; King, 1993). Theories of chromosomal speciation have been revisited recently, but the emphasis has been on the barriers that rearrangements create to recombination between coadapted gene complexes rather than on the reduction in hybrid fertility (Rieseberg, 2001; Rieseberg and Burke, 2001; Navarro and Barton, 2003).
The degree of hybrid sterility is a function of both karyotypic (White, 1957, 1973; Gropp et al., 1982; King, 1993; Noor et al., 2001; Piálek et al., 2001) and genetic ("genic" of Dobzhansky, 1933, 1937; Coyne, 1984) divergence between populations. It is hard to separate the effects of these two components and determine a posteriori which component has been principally responsible for establishing barriers to gene flow between species. In this study, we used genetic divergence to deduce the phylogeny of Agrodiaetus, a species-rich genus of butterflies that shows extreme karyotypic diversity. A reliable phylogeny is a necessary prerequisite to evaluation of the relative roles of genetic and karyotypic divergences for speciation within the genus.
Newly rearranged chromosomes usually occur in heterozygotes and are often associated with heterozygote disadvantage. Therefore, their spread to fixation within a species is considered unlikely. Not surprisingly, many taxa do not undergo extensive karyotypic changes during speciation and tend to be karyotypically conservative at the genus or family level. For example, most species in the order Lepidoptera (Insecta) have karyotypes with 31 pairs of chromosomes (Robinson, 1971; Stekolnikov et al., 2000). This chromosome number arose in the Mesozoic and has remained unchanged in most groups for more than 70 million years (Suomalainen, 1969; Lukhtanov, 2000). Some lepidopteran taxa have distinct modal chromosome numbers, however. Most lycaenid butterflies, for example, have a haploid complement of either 23 or 24 chromosomes (Lesse, 1960b; Robinson, 1971; Lorkovic, 1990; Stekolnikov et al., 2000). Almost every lycaenid with a haploid chromosome number other than 23 or 24 belongs to one of three closely related genera: Agrodiaetus (n = 10–125), Lysandra (n = 24–92), or Plebicula (n = 134–225).
The genus Agrodiaetus Hübner, 1822 is distributed through the western Palearctic, predominantly in the Caucasus, Iran, Turkey, and Central Asia. In the most recent review of the genus, Häuser and Eckweiler (1997) recognized 56 species, 113 subspecies, and 28 well-differentiated taxa of unclear taxonomic status, either species or subspecies. The genus currently includes about 90 valid species when new species described since 1997 are added (Olivier et al., 1999; Carbonell, 2000, 2001; Dantchenko, 2000; Hagen and Eckweiler, 2001; Schurian and Hagen, 2001, 2003; Skala, 2001; Lukhtanov and Dantchenko, 2002a).
Species within Agrodiaetus are extremely uniform and exhibit few differences in characters traditionally used in classification, such as wing pattern and/or aspects of the male and female genitalia. However, these species vary greatly in their karyotypes, ranging from n = 10 (A. posthumus; Lesse, 1959a) to n = 125 (A. dolus; Lesse, 1962b; for karyotype images, see Lukhtanov and Dantchenko, 2002a, 2002b). Karyotypes may provide the only diagnostic characters for some Agrodiaetus species (Lesse, 1960a, 1961; Lukhtanov, 1989; Kandul, 1997; Kandul and Lukhtanov, 1997), and a description of the karyotype has become a necessary requirement for describing new Agrodiaetus species (Lesse, 1957, 1959a, 1959c, 1960a; Lukhtanov et al., 1997; Olivier et al., 1999; Lukhtanov and Dantchenko, 2002a).
Eliot (1973) included the genus Agrodiaetus within the section Polyommatus. Some taxonomists have preferred to consider Agrodiaetus as a subgenus of the large polytypic genus Polyommatus Latreille, 1804 (Dantchenko and Lukhtanov, 1993, 1994; Hesselbarth et al., 1995; Eckweiler and Häuser, 1997; Häuser and Eckweiler, 1997; Lukhtanov et al., 1997, 1998; Koçak and Seven, 1998; Dantchenko, 2000). Different groups within Polyommatus (sensu lato) do not show clear phenotypic differences. Therefore, placements of species within Polyommatus (sensu lato) have changed repeatedly since the comprehensive revisions by Forster (1936, 1938) and Stempffer (1937) and are still disputed (Hesselbarth et al., 1995). This confusion prompted Bálint and Johnson (1997) to abrogate the taxon Agrodiaetus entirely and to consider Polyommatus as a polytypic genus containing many species groups, with the species of Agrodiaetus parceled out into different species groups. Although the monophyly of different groups within Polyommatus (senso lato) and their relationships are questionable, for clarity we have followed Eliot (1973) rather than Bálint and Johnson (1997) and refer to these groups as genera within the Polyommatus section.
Agrodiaetus species are small (wing span 1.8–3 cm) brown or blue butterflies. Female butterflies are always warm brown, whereas males can have either blue or brown wings. In the latter case, they resemble females. Thus, a species can be classified as either dimorphic or monomorphic depending on the color of males. Lesse (1960a) divided the genus into three species complexes based on male coloration and the presence of well-developed tufts formed by androconial scales (Table 1). Forster (1956, 1960, 1961) divided the genus into numerous polytypic species based on geographic distribution and classic morphological characters (wing color patterns and genital structure). The karyotype studies of Lesse (1957, 1959a, 1959c, 1960a, 1960b, 1962b), which appeared concurrently with Forster's revision, revealed that Agrodiaetus species had extremely diverse karyotypes. Furthermore, some of the species in Forster's system appeared to be complexes of sympatric sibling species that could be identified by their sharply differing karyotypes (Lesse, 1957, 1959a, 1959b, 1960a, 1962a). New Agrodiaetus species were described exclusively on the basis of sympatric and temporal cooccurrence of karyotypically distinct "races" (Lesse, 1960a).
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Subsequent analyses of the scarce morphological characters resulted in partitioning of Agrodiaetus into species groups named after their oldest members (Table 1). In the classification of Hesselbarth et al. (1995), Agrodiaetus was divided into eight species groups: actis, admetus, carmon, damon, damone, dolus, poseidon, and transcaspicus. Eckweiler and Häuser (1997) recognized the admetus and dolus groups but argued that available evidence was too weak to support the remaining groups. Instead, they erected a more inclusive damon group that combined the membership of Hesselbarth et al.'s actis, carmon, damon, damone and transcaspicus groups with some species from the poseidon group. The remainder of the poseidon group was renamed the dama group, and three additional species groups, the dagmara, erschoffii (= Paragrodiaetus Rose and Schurian, 1977) and iphigenides groups, were erected to accommodate species from eastern Iran and Central Asia that had not been considered by Hesselbarth et al. (1995).
Although Agrodiaetus has recently attracted interest from taxonomists (Carbonell, 1998, 2000, 2001; Koçak and Seven, 1998; Olivier et al., 1999; Lukhtanov and Dantchenko, 2002b; Rose, 2002), in only one study has the monophyly of Agrodiaetus been tested. Mensi et al. (1994) found Agrodiaetus to be monophyletic, but their study included representatives of only 3 of the 12 species groups and used only a single outgroup from within the section Polyommatus. Therefore, basic questions remain to be resolved regarding the monophyly of Agrodiaetus and evolutionary relationships among its species before the role of karyotype diversification in the radiation of Agrodiaetus can be analyzed.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionAppendix 1AcknowledgmentReferences |
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Agrodiaetus Species Sampling and Choice of Outgroups
We sampled representatives from 10 of the 12 species groups of Agrodiaetus and from 14 related genera within the section Polyommatus (Appendix 1) in an effort to test Agrodiaetus monophyly. Two Agrodiaetus species groups were not included because we were unable to obtain specimens. In particular, species from the dagmara group occur in remote localities of the Pamir region of Tadzhikistan and are extraordinarily difficult to collect. We were also unable to obtain specimens of the dama group. In choosing potential outgroups, we followed Forster (1938), who considered Agriades, Albulina, Aricia, Cyaniris, Eumedonia, Lycaeides, Lysandra, Meleageria, Polyommatus, Plebejus, and Vacciniina to be closely related to Agrodiaetus. Since Forster's time, new genera have been delineated from among these taxa, including Neolysandra, Plebicula, Rimisia, and Sublysandra. We were able to obtain representatives of all these genera except Albulina. Of the taxa sampled in this study, only Aricia, Agriades, Plebejus, and Polyommatus were treated as distinct genera by Bálint and Johnson (1997). The rest of the taxa, including Agrodiaetus, Cyaniris, Lysandra, Meleageria, Neolysandra, Plebicula, and Sublysandra, were designated as synonyms of Polyommatus.
Four Agrodiaetus specimens originally were not identified to the species level. These specimens were assigned to species groups using morphological characters. We were unable to obtain karyotypes for three of these specimens. A karyotype was determined for the specimen VL01L342, but this did not help to identify it to the species level. The specimen was phenotypically close to A. eriwanensis eriwanensis (n = 34) and A. eriwanensis interjectus (n = 29–32; Lesse, 1960a), but its karyotype was different (n = 40–42). In our analysis, A. eriwanensis eriwanensis with n = 34 was a sister species of this specimen. Hesselbarth et al. (1995) placed A. interjectus and A. eriwanensis in the admetus group. According to the recovered phylogeny, A. eriwanensis and the specimen VL01L342 clustered among species from the dolus group (Fig. 1). Unfortunately, A. interjectus was not represented in our analysis. Recently, the specimen VL01L342 was used as a paratype for the newly described A. dantchenkoi Lukhtanov and Weimers 2003 (Lukhtanov et al., 2003). The other three unidentified specimens have been given reference numbers.
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DNA Extraction and Gene Sequencing
Many Agrodiaetus species are difficult to identify accurately without karyotype information. Therefore, testes were extracted from male specimens and fixed for karyotype analysis in freshly prepared Carnoy fixative (100% ethanol and 100% acetic acid, 3:1) before the specimen's body was put into 100% ethanol for DNA preservation. In most cases, the same Agrodiaetus specimen was used for karyotype and phylogenetic analyses. In some cases, we could not obtain karyotypes from collected specimens. In these cases, we used karyotypes previously obtained from the same population (if available) or from a different population of the same taxon. The specimens used in this study and the source of karyotype data are listed in Appendix 1. All specimens are deposited in the DNA and Tissues collection of the Museum of Comparative Zoology (Harvard University, Cambridge, MA).
Four abdominal segments were used for DNA extraction. The segments were homogenized in 2% SDS buffer and digested with proteinase K (20 mg/ml) for at least 3 hr at 60°C. DNA was purified through successive ethanol precipitations and stored in Tris-EDTA buffer (pH 8.0) at –20°C. Two mitochondrial genes, cytochrome oxidase subunit I (COI) and cytochrome oxidase subunit II (COII), and a nuclear gene, elongation factor 1- (EF1-), were used to reconstruct the phylogeny of Agrodiaetus. The primers used for the mitochondrial DNA (mtDNA) amplification have been described (Rand et al., 1999; Monteiro and Pierce, 2001). PCRs (50 µl) were carried out in the DNA Engine thermal cycler (MJ Research) and typically contained 0.5 µM of each primer, 0.8 mM dNTPs, Qiagen (Valencia, CA) PCR buffer with additional MgCl2 to a final concentration of 2 mM, and 1.25 units Qiagen Taq DNA polymerase. The typical thermal profile was 37 cycles of 95°C for 60 sec, 47°C for 60 sec and 72°C for 90 sec. Touchdown PCR with a starting annealing temperature of 53°C and Qiagen Q solution was used to amplify three fragments of EF1- with primers listed in Table 2. After amplification, the double-stranded DNA was purified using QIAquick PCR purification kits (Qiagen) prior to direct sequencing. Cycle sequencing reactions (10 µl) were performed using ABI Prism Big Dye 2 terminator cycle sequencing kits (Applied Biosystems, CA). Both strands of the PCR product were sequenced in a 3100 Genetic Analyzer (Applied Biosystems/Hitachi).
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Phylogenetic Analysis
Sequence alignments and characteristics
Mitochondrial and nuclear sequences were edited and aligned against the total mtDNA sequence (GenBank NC_002355 [GenBank] ; Lee et al., 1999) and the EF1-
sequence (GenBank, D13338
[GenBank]
; Kamiie et al., 1993) of Bombyx mori L. using Sequencher 3.1 (Genecodes Corporation, Ann Arbor, MI). Alignments were unambiguous for both mitochondrial and nuclear gene fragments. All fragments of translated genes were of equal length except the COII sequences of Eumedonia persephatta minshelkensis and Vacciniina fergana, which had a triplet insertion in the same position. This insertion was excluded from further phylogenetic analysis. Primer sequences were cropped, and missing data and ambiguities were designated by the letter "N." All sequences were submitted to GenBank (AY496709
[GenBank]
–AY496850
[GenBank]
).
The 113 continuous sequences of COI, tRNA-leu, and COII genes were aligned in a data set that was partitioned into respective genes using PAUP* 4.0b10 (Swofford, 2000). The tRNA-leu gene was excluded from further phylogenetic analysis. Additional EF1- sequences were obtained for a representative of each outgroup genus (Appendix 1) and each major Agrodiaetus clade recovered in our analysis (Fig. 1). EF1- was also sequenced from A. erschoffii and A. glaucias because Rose and Schurian (1977) had segregated these species into a distinct genus, Paragrodiaetus. Separate sequences of COI, COII, and EF1- genes were concatenated into a single partitioned data set in MacClade (Maddison and Maddison, 1992). The character statistics were presented separately for total, ingroup, and outgroup sequences from each data set in Table 3. Base frequency homogeneity was tested separately for every data set with the 
2-test in PAUP*. Because only informative sites can affect the phylogenetic analysis, uninformative sites were excluded from these tests.
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We used a partition homogeneity test (incongruence length difference [ILD]; Farris et al., 1995) to acquire preliminary data on the homogeneity of our data sets. ILD tests were performed in PAUP* using heuristic searches with tree bisection–reconnection (TBR) branch swapping and 100 random taxon addition replicates, saving no more than 100 equally parsimonious trees per replicate. Recent studies (Cunningham, 1997; Barker and Lutzoni, 2002; Darlu and Lecointre, 2002; Dolphin et al., 2002; Dowton and Austin, 2002) have cast doubt on the utility of the ILD test for detecting incongruence between different data sets caused by differences in evolutionary constraints and/or tree topologies. Therefore, we used additional data to make a final decision whether to combine data sets or analyze them separately.
Maximum parsimony
Maximum parsimony (MP) analysis was conducted with PAUP*. A heuristic search was performed with TBR branch swapping and 1,000 random taxon addition replicates, saving no more than 100 equally parsimonious trees per replicate. In addition, PAUPRat (Sikes and Lewis, 2001) was used for MP analysis of the large COI + COII data set because of its speed in searching large data sets (Nixon et al., 1998; Nixon, 1999). During 200 iterations in PAUPRat, 10%, 15%, and 20% of equally weighted characters were perturbed. Tree lengths reported in this study included parsimony-uninformative characters. To estimate branch support on the recovered topology, nonparametric bootstrap (bt) values (Felsenstein, 1985) and Bremer support (Br; Bremer, 1994; for the discussion on the interpretation of Bremer support, see DeBry, 2001) were assessed with PAUP* and Autodecay 4.0.2' (Eriksson, 1998), respectively. One thousand bootstrap pseudoreplicates were analyzed under a heuristic search with TBR branch swapping and 100 random taxon addition replicates, saving no more than 100 equally parsimonious trees per replicate. The same settings for the heuristic search were used to estimate Bremer support.
Substitution models
The general time reversible model with invariant sites and gamma distribution (GTR+I+
) was the substitution model selected for the COI + COII data sets (113 and 29 sequences, respectively) by both hierarchical likelihood ratio tests (hLRTs; Huelsenbeck and Rannala, 1997) and the Akaike information criterion (AIC; Akaike, 1974) as implemented in Modeltest 3.06 (Posada and Crandall, 1998). The application of Modeltest 3.06 to the EF1- data set resulted in two different substitution models. The Tamura and Nei (1993) model with invariable sites and gamma distribution (TrNef+I+) was chosen by hLRTs, and the GTR+I+ model was chosen by AIC. The GTR+I+ model was chosen for the combined COI + COII + EF1- data set by both hLRTs and the AIC.
Bayesian inference
Bayesian analysis was done in a likelihood framework as implemented by MrBayes 2.01 (Huelsenbeck, 2000; Huelsenbeck and Ronquist, 2001) with uninformative priors. Multiple Bayesian searches using Metropolis-coupled Markov chain Monte Carlo sampling were conducted. One cold and three heated Markov chains, applying MrBayes default heating values (t = 0.2), were used in the analysis. Because the TrNef+I+ model is not implemented in MrBayes 2.01, the GTR+I+ model was used for every data set. Model parameter values were treated as unknown and were estimated in each analysis. To ensure that our Bayesian analyses were not trapped in local optima, each analysis was run four times, starting from different random trees, and average log-likelihood (lnL) values (±SD) at stationarity were calculated with Microsoft Excel and compared for convergence. Each Bayesian analysis was run for 1,000,000 generations, with trees sampled every 100 generations. The number of sampled trees to be discarded as representing the burn-in period was determined graphically. To estimate posterior probabilities (pP) of recovered branches (Larget and Simon, 1999; Huelsenbeck et al., 2001), 70% majority rule consensus trees were created from the remaining trees using PAUP*. Phylograms were created as average-branch-length consensus trees with MrBayes.
Dating phylogenetic events
To test the homogeneity of substitution rates (for the molecular clock hypothesis) across the average-branch-length consensus of Bayesian trees recovered for the larger COI + COII data set, we applied the LRT (Huelsenbeck and Rannala, 1997) with and without the enforcement of a molecular clock. Likelihood scores of the ingroup topology (Fig. 1) were assessed under the GTR+I+ model as implemented in PAUP*. The LRT revealed a significant deviation from rate consistency (P < 0.001) across different branches. Therefore, a nonparametric rate smoothing (NPRS) algorithm (Sanderson, 1997), implemented in TreeEdit 1.0 (Rambaut and Charleston, 2002), was used to homogenize evolutionary rates across the topology. Mean uncorrected pairwise distances, calculated in MEGA2 (Kumar et al., 2001), were used to calibrate the smoothed topology.
Character-state reconstruction
MacClade 4.0 (Maddison and Maddison, 1992) was used to reconstruct the ancestral karyotype for the Agrodiaetus lineage. To decrease potential bias of certain chromosome numbers, only single individuals of every sampled Agrodiaetus species with known chromosome numbers was used. Thus, mitochondrial sequences of 71 distinct species were used to build a topology for the tracing of karyotype changes. The strict consensus was built from 56 recovered MP trees: total length = 2,144; consistency index (CI) = 0.392; retention index (RI) = 0.627. Haploid chromosome numbers (n) were arbitrarily coded as 12 ordered character states and mapped on the recovered phylogeny. The original PAUP* and MacClade files are available from the authors.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionAppendix 1AcknowledgmentReferences |
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Analysis of the COI + COII Data Set
The alignment of 113 COI + COII sequences recovered 513 parsimony-informative characters (26% of 1,969 sites; 25.8% from COI and 26.4% from COII; Table 3). The application of the
2-test in PAUP* did not reject the hypothesis of homogeneity of nucleotide frequencies for parsimony-informative characters in every pair of taxa (P = 0.99). Estimates of substitution rates in COI and COII were similar for first and third codon positions, as reflected by the percentage of parsimony-informative characters. By contrast, the substitution rate for the second codon position in the COII gene (1.7%) was twice as high as that for the COI gene (0.9%, Table 3). The frequency of adenine (A) and thymine (T) was slightly higher for outgroup sequences in every data set presented in Table 3. This higher frequency of A + T can be explained by the older age of the outgroup species and their concomitant higher saturation levels at the third codon position. The COII gene fragment had a higher saturation rate of A + T (0.7730 ± 0.0.006) than did the COI gene fragment (0.7212 ± 0.007, Table 3). High levels of A + T saturation in mitochondrial sequences (> 75%) are well known for insects, especially at third codon positions (Crozier et al., 1989; Pedersen, 2002). The relative increase of A + T saturation between COII and COI was greater for first and second codon positions (6.6%) than for third codon positions only (2.6%). Comparing the sequenced genomes of Apis mellifera and Drosophila melanogaster, Crozier and Crozier (1993) reported that COII had a higher substitution rate than did COI in insects. This difference in substitution rate could contribute to the higher A + T saturation for COII.
The ILD test between full-length sequences of COI and COII revealed no significant conflict (P = 0.14). However, when the ILD test was performed for two and three equal size fragments of COI and COII, following the suggestion of Dowton and Austin (2002), significant phylogenetic conflict was revealed (P = 0.01). Significant phylogenetic conflict (P = 0.01) also was found between equal size fragments from within the COI gene. Given that (1) mitochondrial genes usually do not recombine (for review, see Eyre-Walker and Awadalla, 2001) and (2) actively translated mitochondrial genes evolve under similar conditions, these results were unexpected. However, the ILD test is particularly prone to type I errors (rejecting the true hypothesis; Darlu and Lecointre, 2002), different levels of homoplasy and phylogenetic noise between large data sets per se can also cause significant conflict (Dolphin et al., 2002) and the heterogeneity of evolutionary rates of data sets can make results of the ILD test misleading (Barker and Lutzoni, 2002). Therefore, we questioned the results of the ILD tests that detected incongruence between equalsize fragments of COI and COII, and we combined the two gene fragments in our analysis.
The ratio of transitions to transversions (Ti/Tv) in third codon positions was plotted against the number of transversions in third codon positions to assess the degree of saturation due to multiple transitions in third codon positions. The Ti/Tv ratio expected at saturation (following Holmquist, 1983) was not reached (0.166; plot not shown). A partition homogeneity test (ILD) for the combined data set of COI + COII did not reveal significant conflict between first and second versus third codon positions (P = 0.4). Therefore, we assigned equal weight to transitions and transversions for all sites in the analysis. We also performed MP analysis using various alternative weighting schemes. These gave the same strict consensus tree, except that the weakly supported clade VII became paraphyletic in some analyses (data not presented).
Heuristic search with unlimited number of trees saved per replicate in PAUP* resulted in 7,433 MP trees of TL = 2,508, CI = 0.356, and RI = 0.719 (Figs. 1, 2). Three independent trials using perturbations of 10%, 15%, and 20% of parsimony-informative characters during 200 iterations in PAUPRat yielded 176, 140, and 125 MP trees, respectively, of TL = 2508, CI = 0.356, and RI = 0.719. The strict consensus of 441 MP trees recovered by PAUPRat had the same topology as the strict consensus of 7,433 MP trees inferred by the heuristic search (LRT under the GTR+I+: –lnL1 = 16076.516 and –lnL2 = 16059.662; 2111 = 33.7, P = 1.0).
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Four independent Bayesian analyses converged on statistically equivalent log-likelihood scores and reached stationarity at no later than 200,000 generations (plots not shown). Majority consensus trees of four rounds of Bayesian analysis were identical, and the posterior probability values supporting congruent nodes were highly correlated (not shown), providing support for the assumption that the analyses converged on a single optimum. The majority consensus trees are presented in Figures 1 and 2. Figure 1 illustrates relationships within Agrodiaetus, whereas Figure 2 presents relationships between Agrodiaetus and the outgroups.
The monophyly of Agrodiaetus is well supported by both MP (bt = 94%; Br = 4) and Bayesian inference (BI) analyses (pP = 1.00) (Figs. 1, 2). Although the MP and BI trees are similar, there are some differences at both the ingroup and outgroup levels. The most conspicuous difference is the position of clades IV + V and VI + VII + VII within Agrodiaetus (Fig. 1). However, the placement of these clades on the MP tree was poorly supported (bt < 50 and Br = 1). The use of site-specific substitution rates for different codon positions (instead of substitution rates under the gamma distribution) with the GTR+I model did not substantially change the inferred BI tree, although clade XII appeared paraphyletic (Fig. 1). According to the MP analysis, the genus Polyommatus is sister to Agrodiaetus, whereas under the Bayesian analysis, Polyommatus thersites and the clade ((P. icarus, P. amandus) Meleageria daphnis, Plebicula dorylas) forms an unresolved trichotomy with Agrodiaetus (Fig. 2).
COI + COII sequences provided considerable resolution within Agrodiaetus. The alignment of 91 ingroup sequences recovered 356 parsimony-informative characters (18% of sites). Constraints corresponding to the proposed systems (Table 1) of Hesselbarth et al. (1995) and Eckweiler and Häuser (1997) were enforced during a heuristic search in PAUP* to test current hypotheses of Agrodiaetus classification. Likelihood scores of the strict consensus trees of the resultant MP trees were calculated in PAUP* under GTR+I+. These analyses yielded –lnL = 17268.669 for Hesselbarth et al. (1995), –lnL = 17241.007 for Eckweiler and Häuser (1997), and –lnL = 16059.662 without any constraints. Thus, both prior taxonomic hypotheses were rejected by a likelihood ratio test (2111 = 2418.014 and 2111 = 2362.69, respectively; P = 0.0000).
Our Agrodiaetus specimens could be divided into 12 clades (I–XII, Fig. 1). Each clade was recovered by both MP and BI methods except clade VII, which was monophyletic in all MP trees but paraphyletic on the BI tree (Fig. 1). Each of the clades I, III, VI, X, and XI contained members of a single species group. Three subspecies of A. damon form clade I (bt = 100; Br = 19, pP = 1.00; Fig. 1) at the base of the Agrodiaetus clade, whereas the remainder of the damon group are placed in clades IV, V, and IX. Most members of the admetus group are found in clade III (with the remainder in clade II). Most members of the dolus group are clustered together in clade II, but two species, A. antidolus and A. kurdistanicus, together form clade VI (Fig. 1). The three representatives of the poseidon group in our sample grouped together as clade X (bt = 76; Br = 3; pP = 1.00; Fig. 1). Most members of the actis group are found in clade XI (with the remainder in clade XII).
Our analysis recovered substantial structure among Agrodiaetus clades, with broad agreement between MP and BI trees (Fig. 1). Clade I was recovered as basal to the rest of Agrodiaetus (clades II–XII) in both analyses. The larger of these clades had bt < 50, Br = 2, and pP = 0.94. Within this clade, both analyses recovered four subclades: (II, III), (IV, V), (VI, VII, VIII), and (IX, ((X, XI), XII)), with ((X, XI), XII) particularly well supported. MP and BI analyses differed as to the relationships among these subclades.
The hypothesis of phylogenetic independence between chromosome numbers and the recovered ingroup topology was tested using the test for serial independence (TFSI; Abouheif, 1999) implemented in Phylogenetic Independence 2.0 (Reeve and Abouheif, 2003). Only single representatives of Agrodiaetus species with known chromosome numbers were used in this test. The test rejected the hypothesis that chromosome numbers were not correlated with phylogeny (P = 0.0001).
Analysis of the COI + COII + EF1- Data Set
EF1- has provided substantial resolution and support for recovered branches at the species and genus levels among Lepidoptera (Cho et al., 1995; Monteiro and Pierce, 2001). However, in our analysis, 29 EF1- sequences of a 1,195-bp region provided only 88 parsimony-informative characters (7.4% of all sites; Table 3). The alignment of 14 ingroup sequences of Agrodiaetus provided only 19 parsimony-informative characters (1.6% of total sites), whereas 15 outgroup sequences recovered 54 parsimony-informative characters. The application of the 2-test in PAUP* did not reject the hypothesis of homogeneity of nucleotide frequencies for parsimony-informative characters in every pair of taxa (P = 0.87).
Transition and transversion substitutions were treated equally at every site during MP analysis. A partition homogeneity test (ILD) conducted under a heuristic search in PAUP* revealed a significant conflict (P = 0.04) between the phylogenetic signals of COI + COII combined and EF1- sequences and between equal size fragments (1,195 bp) of COI and EF1- (P = 0.02). However, the P value recovered in both tests was above the critical value (P = 0.01–0.001) suggested by Cunningham (1997) in testing for incongruence between different data sets. Therefore, we chose to perform separate and combined analyses of these gene fragments.
A heuristic search with equally weighted characters yielded 6,629 MP trees for the EF1- data set (TL = 354; CI = 0.678; RI = 0.562). Four independent Bayesian analyses converged on statistically equivalent log-likelihood scores and reach stationarity at no later than 50,000 generation (plots not shown). The constructed majority consensus trees were identical, whereas posterior probabilities of congruent branches were almost equal. The strict consensus of 6,629 MP trees and a 70% consensus of trees sampled in Bayesian analyses are shown in the Figure 3. According to both the MP and BI methods, the monophyly of Agrodiaetus was well supported (bt = 90; Br = 4; pP = 1.00). Bayesian analysis of the EF1- data set provided better resolution and support than the MP analysis at the outgroup level (Fig. 3). According to the BI majority tree, Lysandra bellargus and Meleageria daphnis form a clade (pP = 0.80), which is the sister lineage to Agrodiaetus. The clade containing these three taxa has pP = 0.73 (Fig. 3). Three clades comprising (1) Polyommatus amandus + P. icarus + Sublysandra cinyraea; (2) Lysandra bellargus + Meleageria daphnis; and (3) Plebicula dorylas formed a polytomy with Agrodiaetus on the BI phylogram (figure not shown). Lycaeides argyrognomon + Plebejus argus formed a clade on both the MP and BI trees (bt = 56; Br = 1; pP = 0.99).
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The alignment of the 29 COI + COII sequences recovered 327 parsimony-informative characters (16.6% of the total sites; Table 3). A
2-test in PAUP* rejected the hypothesis of homogeneity of nucleotide frequencies for parsimony-informative characters (P = 0.0002). There is little evidence that heterogeneity of nucleotide composition leads to significant phylogenetic error (Rosenberg and Kumar, 2003). However, in an attempt to rule out the possible effect of heterogeneity on tree inference, we performed a LogDet (LD) distance transformation (Lockhart et al., 1994) while accounting for invariable sites (LD+I) on the 29 COI + COII sequences in PAUP*. The LD distance transformation was specifically designed to account for a strong bias in nucleotide frequencies among sequences (Lockhart et al., 1994). A heuristic search for a minimum evolution objective function was used to infer distance trees from the DNA distances calculated under LD+I transformation and the GTR+I+ model in PAUP*. The null hypothesis of no difference between the two trees was not rejected by the Kishino–Hasegawa parametric test (P = 0.5128; Kishino and Hasegawa, 1989) or by the Templeton nonparametric test (P = 0.5127; Templeton, 1983). The distance trees built with Tajima–Nei distances (Tajima and Nei, 1982) and Kimura two-parameter distances (Kimura, 1980) were identical to the tree built with LD+I distances (TL = 1,873; CI = 0.513; RI = 0.431; –
nL = 14075.3125 under the GTR+I+ model). Therefore, we carried out our phylogenetic analysis without accounting for the heterogeneity of nucleotide frequencies.
A heuristic search performed in PAUP* yielded 22 MP trees of TL = 1,470, CI = 0.485, and RI = 0.437 for the 29 COI + COII sequences. Although the MP trees inferred from separate phylogenetic analysis of COI + COII and EF1- differed in branching order at both the ingroup and outgroup level, these differences were not well supported (Fig. 4). The group Lycaeides argyrognomon + Plebejus argus is well supported in both of these MP trees (Fig. 4). The main relationships among Agrodiaetus clades inferred from the large data set of COI + COII (113 sequences) were also recovered using the small data set of COI + COII (29 sequences). In particular, subclades (II, III), (IV, V), (VI, VII, VIII), and ((X, XI), XII) (Figs. 1 and 4) were supported from analyses of both data sets.
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The analysis of the combined data matrix of COI + COII + EF1-
(29 sequences) resulted in well-supported MP and BI trees. A heuristic search recovered 12 MP trees of TL = 1,852, CI = 0.515, and RI = 0.440. Four independent Bayesian analyses converged on statistically equivalent log-likelihood scores and reached stationarity at no later than 60,000 generation (plots not shown). The constructed majority consensus trees are identical, and posterior probabilities of congruent branches are almost equal. The monophyly of Agrodiaetus was again recovered with bt = 97, Br = 7, and pP = 1.00 (Fig. 4). According to the strict consensus of 12 MP trees, Polyommatus icarus and P. amandus form a sister clade to Agrodiaetus (bt < 50; Br = 1), whereas P. icarus is the sister to Agrodiaetus on the average-branch-length consensus of sampled Bayesian trees (topology not shown). A more inclusive group is formed by Agrodiaetus, Polyommatus, and Plebicula dorylas + Meleageria daphnis (bt = 51; Br = 2; pP = 1.00; Fig. 4). This group together with Neolysandra alexander and Lysandra bellargus + Sublysandra cinyraea form a clade with bt = 91, Br = 9, and pP = 1.00. The MP and BI trees differ in the position of Rimisia miris and Eumedonia persephatta (Fig. 4). However, the position of Rimisia miris on the MP tree is weakly supported (bt < 50, Br = 0). The clade Lycaeides argyrognomon + Plebejus argus was recovered on both the MP and BI trees (bt = 100; Br = 18; pP = 1.00; Fig. 4). The BI tree has better resolution than the MP tree at the outgroup level. Thus, Vacciniina fergana and Agriades pheretiades are the taxa most distant from Agrodiaetus, followed by Rimisia miris and Cyaniris semiargus.
Dating Main Phylogenetic Events
To calibrate a molecular clock, a branching point on a tree must be linked to a particular geological event so that substitution rate can be scaled in evolutionary time. Unfortunately, our data are not amenable to such an exercise because lycaenid butterflies lack a useable fossil record. Additionally, our current sample does not include a phylogeographic event that could be independently dated. Therefore, we used two different published estimates of the mtDNA substitution rate in arthropods. The substitution rate of COI sequence in Tetraopes beetles (Farrell, 2001) provided a slower estimate of 1.5% uncorrected pairwise distance per million years and hence older estimated ages. The substitution rate for the entire mtDNA genome of various arthropod taxa (Brower, 1994) provided a faster estimate of 2.3% uncorrected pairwise distance per million years and hence younger estimated ages. The use of two different rate estimations to calibrate the recovered tree was intended to correct partially for the potential bias generated by not taking into account the sequence divergence already presented in the ancestral population at the time of its divergence into evolutionary distinct lineages.
The Bayesian topology of average-branch-length consensus reconstructed from the data set of 113 COI + COII sequences was used for dating phylogenetic events. Heterogeneous substitution rates across the topology were homogenized with the NPRS algorithm (Sanderson, 1997). We decided against using the basal node to estimate the age of Agrodiaetus because multiple substitutions and hence the error of estimation for pairwise distances would increase with overall sequence divergence (Hillis et al., 1996). Thus, the topology was calibrated using mean uncorrected pairwise distances between clade XII and clade X + XI (Fig. 5). This node (point 1, Fig. 5) was chosen because it is well supported and contains few clades. Given the substitution rate of 2.3% or 1.5% per million years, this point corresponded to 1.23–1.89 million year ago (MYA). Applying this calibration to the recovered topology, the origin of the genus Agrodiaetus was dated to 2.51–3.85 MYA. Figure 5 presents two dating scales applied to the Bayesian topology of average-branch-length consensus smoothed with the NPRS algorithm.
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Agrodiaetus: Monophyly and Ingroup Relationships
Phenotypic differences among species within the genus Agrodiaetus are slight, with only one superficial character, a long white streak on the underside of the hind wings, considered a possible synapomorphy. However, this character is variable and can be undeveloped or absent in some individuals, populations, and even species. Furthermore, a white streak of similar shape and position is found in some species of Polyommatus (sensu stricto) and Aricia. Extreme karyotypic diversity also is not restricted to Agrodiaetus. In particular, two other genera within the section Polyommatus, Lysandra (n = 24–92) and Plebicula (n = 131–225), have marked variation in chromosome number. Thus, Agrodiaetus has been a problematic taxon because of the absence of characters that uniquely separate it from other genera in the section.
Gene trees inferred from both mitochondrial and nuclear gene fragments, using both MP and Bayesian methods, all support monophyly of Agrodiaetus (Figs. 2–4). The fact that differently inherited genes gave concordant results increases the probability that the genus, as currently defined, is indeed monophyletic. The sister taxon to Agrodiaetus is likely to include species within the genus Polyommatus (sensu stricto). In our study representatives of this genus emerged close to Agrodiaetus on every MP tree inferred from separate and combined analyses of mitochondrial and nuclear genes and appeared as part of a basal polytomy in most Bayesian trees. The exception was the BI majority tree inferred from EF1- (Fig. 3). This placed (Lysandra bellargus, Meleageria daphnis) as the sister taxon of Agrodiaetus. The clade (Agrodiaetus, (Lysandra bellargus, Meleageria daphnis)), however, had weak support (pP = 0.73) and did not appear in any other tree. Thus, all four phylograms reconstructed as average-branch-length consensus trees from four independent rounds of Bayesian analysis of the EF1- data set did not contain this clade. Instead, they had a polytomy with four clades: (1) Agrodiaetus species; (2) Polyommatus amandus, P. icarus, and Sublysandra cinyraea; (3) Lysandra bellargus and Meleageria daphnis; and (4) Plebicula dorylas (not shown).
Our analysis identified 12 major clades within Agrodiaetus (Figs. 1, 5), labeled I–XII to facilitate discussion. Of these, clade VII is the most weakly supported. At this stage of our study, we have not thoroughly sampled some species groups. In particular, our representatives of the A. admetus group were biased toward species with high chromosome numbers, whereas the important species A. alcestis (n = 19 or 20; Lesse, 1960a) and A. interjectus (n = 29–31; Lesse, 1960a) were not included. Although our analysis recovered a monophyletic poseidon group, difficult logistics (A. deedi occurs in Lebanon, where we have not yet collected) and the rarity of specimens prevented us from obtaining three other species considered part of the poseidon group (sensu Hesselbarth et al., 1995).
In previous classifications (Lesse, 1960a; Hesselbarth et al., 1995; Eckweiler and Häuser, 1997; Table 1), the admetus group contained exclusively monomorphic butterflies (both sexes brown), the dolus and erschoffii groups contained both monomorphic and dimorphic species, and the other species groups contained only dimorphic species. Mensi et al. (1994) argued that monomorphy is a derived character within Agrodiaetus. Bálint and Johnson (1997) proposed that monomorphy (= discoloration) had multiple origins within the section Polyommatus. Our analysis provides evidence for at least two origins of monomorphy within Agrodiaetus. Most representatives of clade (II, III) are monomorphic, and our trees suggest that monomorphy may be ancestral for this clade, with several of apparent reversals. Monomorphy appears to have evolved independently in A. mithridates, placed in the distant clade XII (Fig. 5).
Rose and Schurian (1977) proposed that the species within the erschoffii group be placed within a separate genus Paragrodiaetus. On the basis of its name, Paragrodiaetus appears to have been considered the sister genus to Agrodiaetus. The two representatives of "Paragrodiaetus" in our analysis (A. erschoffii and A. glaucias) were recovered within clade IX (bt = 97; Br = 8; pP = 1.00; Fig. 1), a clade that also contained members of the A. damon group. Recognition of Paragrodiaetus would render Agrodiaetus paraphyletic.
Mensi et al. (1994) placed the damon group as the basal taxon of Agrodiaetus (only the admetus, dolus, and damon groups were sampled). Our clade I contains all the members of the group studied by Mensi et al. (1994), and consistent with their conclusion, clade I diverges at the base of Agrodiaetus in our analysis. However, other members of the damon group (sensu Hesselbarth et al., 1995) are placed in clades IV, V, and IX, rendering the damon group polyphyletic (Fig. 1). All other species groups recognized by Hesselbarth et al. (1995) are nonmonophyletic in our analysis, except the poseidon group (clade X), which is represented by three species.
Representatives of the same nominal species do not form a monophyletic group in three places in our inferred phylogeny. In clade III, A. demavendi (n = 66–72) occurs in the midst of individuals assigned to A. ripartii (n = 90). In clade VIII, individuals assigned to A. huberti (n = 33–37) and A. turcicolis (n = 19 or 20) are intermixed with each other and with individuals assigned to A. ninae (n = 33 or 34), A. elbursicus (n = 18), and A. aserbeidschanus (karyotype unknown). In clade XII, individuals assigned to A. altivagans (n = 20–22) are intermixed with individuals assigned to several other species (n = 16–26), including A. damocles (n = 25 or 26), which itself is nonmonophyletic. Leaving aside the possibility of mistaken identity, such nonmonophyletic "species" could be explained in multiple ways. All species could be reciprocally monophyletic, with these placements simple artifacts of the limited number of informative characters. Some species could retain ancestral polymorphisms that predate speciation events and thus appear paraphyletic in relation to derived species. Species boundaries could be too broad, with some nominal species containing multiple species that do not form a clade. Species boundaries could be too narrow, with continuing interbreeding between nominal species, in some cases despite differences in chromosome numbers. The available data do not allow us to discriminate among these alternatives.
Age of Agrodiaetus
The ages that we estimate for the origin of major clades are broadly consistent with those inferred from electrophoretic phenotypes by Mensi et al. (1994). Thus, we date the origin of Agrodiaetus around 2.51–3.85 MYA (Fig. 5; node 2), whereas Mensi et al. placed the origin of the genus at 3.1 MYA. Similarly, Mensi et al. estimated the origin of the dolus + admetus groups at 2 MYA, whereas we date the origin of clade II + III, containing species from these groups, at around 1.86–2.85 MYA (Fig. 5; node 3). Mensi et al. estimated the origin of the dolus group at 1 MYA, a date that falls close to our estimate of 1.24–1.91 MYA for the origin of clade II (Fig. 5; node 4), which includes four species from the dolus group and three species from the admetus group.
Three nuclear genes that have been useful for reconstruction of species-level phylogenies in Lepidoptera, 28S ribosomal DNA (rDNA; Miller et al., 1997), wingless (Brower and DeSalle, 1998; Campbell et al., 2000), and EF1- (Cho et al., 1995; Brower and DeSalle, 1998; Monteiro and Pierce, 2001), failed to provide enough informative characters to infer relationships within Agrodiaetus (data for 28S rDNA and wingless not presented). Lineage sorting among species is likely to be a problem for nuclear markers in Agrodiaetus because the time until coalescence for alleles within lineages may be greater than the time between successive speciation events. EF1- failed to provide sufficient phylogenetic signal to resolve relationships among karyotypically well-defined Agrodiaetus taxa, whereas mitochondrial genes yielded a high number of parsimony-informative characters (Table 3). Given that the substitution rate for mitochondrial genes can be four times that for nuclear genes (Page and Holmes, 1998:127), we conclude that species of Agrodiaetus are younger than lepidopteran species for which both mitochondrial and nuclear genes (COI/COII and EF1-, respectively) provide useful phylogenetic signal.
Karyotypic Changes within Agrodiaetus
Three genera in the section Polyommatus exhibit marked karyotype diversity; our research here has focused on relationships within Agrodiaetus. The other two genera, Lysandra and Plebicula, were represented in our analysis as outgroups. The three genera did not cluster together in any of our trees (Figs. 1–4), although Lysandra bellargus, L. caucasica, and L. coridon form a well-supported clade in the MP and BI trees inferred from COI + COII sequences (Fig. 2). Thus, our analysis suggests that accelerated rates of karyotypic diversification have at least three origins within the section Polyommatus (Fig. 6). Further sampling of Lysandra and Plebicula and the inclusion of representatives of currently unsampled genera in the section Polyommatus will further resolve this question.
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The test for serial independence (TFSI; Abouheif, 1999) rejected the hypothesis that chromosome numbers were not correlated with phylogeny (P = 0.0001). This result is not surprising. Casual inspection of Figure 1 reveals that closely related individuals tend to have similar chromosome numbers, although there are some exceptions where sister taxa have very different numbers. To estimate visually a suggested correlation between molecular and karyotypic divergences among Agrodiaetus species, we plotted total pairwise distances (under the GTR+I+
model of DNA substitution) against normalized absolute pairwise differences of chromosome numbers (|nI–nJ|/24) using Microsoft Excel 2001 (Fig. 7). This plot showed a weak positive correlation (R2 = 0.136) between molecular and karyotypic divergences.
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Haploid chromosome numbers ranging from 41 to 50 were recovered as the ancestral state of karyotype for the genus Agrodiaetus under every resolving option for character-state reconstruction in MacClade 4.0 (Maddison and Maddison, 1992): MP, accelerated transformation (ACCTRAN), and delayed transformation (DELTRAN). The recovered ancestral state of karyotype is twice as large as the modal chromosome number of the family Lycaenidae (n = 23 or 24). The closest outgroup species from the genus Polyommatus (P. icarus, P. amandus, and P. thersites) have this chromosome number (n = 23 or 24), as does the ingroup species A. turcicus (n = 24). Only when chromosome numbers were coded as unordered character states were ancestral chromosome numbers (n = 22–25) similar to n = 23 or 24 uncovered in MacClade using DELTRAN, whereas MP and ACCTRAN did not recover an unequivocal ancestral character state.
Setting aside questions about the accuracy of the recovered phylogeny and the subjectivity of character coding, we have little confidence that n = 41–50 was the ancestral condition of Agrodiaetus. Algorithms of ancestral state reconstruction implemented in MacClade are based on parsimony criteria. However, parsimony ignores information about branch lengths. Furthermore, it reconstructs rapidly evolving characters with less accuracy (Felsenstein, 1973; Frumhoff and Reeve, 1994; Schluter et al., 1997) and does not provide the degree of support for its reconstruction (Cunningham, 1999). Perhaps the most distinctive feature of the genus Agrodiaetus is its rapidly evolving karyotype. Therefore, we think that parsimony may provide an inaccurate or even misleading method of ancestral state reconstruction of karyotypes in this group. To our knowledge, available likelihood programs of character reconstruction do not support a simple model of karyotype evolution in Agrodiaetus. Further research will be necessary to reconstruct the ancestral karyotype of Agrodiaetus: additional species of Agrodiaetus with distinct karyotypes must be studied, and additional characters of the karyotype (e.g., its structure, including the relative size of bivalents) must be considered to distinguish common ancestry (= homology) from convergence in chromosome number (= homoplasy).
Whether the ancestral chromosome number was 40–50 (as recovered by parsimony) or 23 or 24 (as inferred by comparative analysis with outgroup species), chromosome numbers have both increased and decreased within Agrodiaetus. The lowest number we observed was in A. posthumus biruni (n = 10 or 11), and the highest was in A. dolus vittatus (n = 123–125) (Appendix 1). The mapping of chromosome numbers onto our phylogeny does not allow simple generalizations (Fig. 1). Clade IV (n = 12–28) and clade XII (n = 16–25) have mostly low numbers, clade III (n = 66–90) has mostly high numbers, and clade IX includes among its members A. phyllis (n = 79–81) and A. posthumus biruni (n = 10 or 11) as sister taxa. This relationship is perhaps the most dramatic example of rapid karyotypic change present in our phylogeny because these sister taxa are estimated to have diverged as recently as 113,000–174,000 years ago (Fig. 5, node 6). This example should be corroborated by further karyotypic studies because the specimens VL01B205 and VL01B206 of A. phyllis and A. posthumus biruni were not karyotyped. An even greater range of chromosome numbers is found in clade II (n = 28–124), but all members of clade V have very similar karyotypes (n = 66 or 67).
The main conclusion stands: chromosome numbers have undergone rapid diversification in the genus Agrodiaetus over the past 3.85 million years, while phenotypic characters have undergone relatively little change. Furthermore, there is evidence that the rate of karyotypic change has varied among lineages within the genus. Thus, we estimate that the roughly fourfold range of chromosome numbers (n = 28–125) found among the six identified species of clade II has arisen in < 1.91 million years (Fig. 5, node 4). By contrast, the five species from clade V for which we obtained karyotypes have similar chromosome numbers (n = 66 or 67) despite the fact that estimated divergence times for this clade are similar to those of clade II (1.22–1.87 and 1.24–1.91 MYA, respectively).
Karyotypic Diversification and Speciation in Agrodiaetus
The occurrence of extensive karyotype diversity among species with little or no genetic and morphological divergence implies the possibility of chromosomal speciation (White, 1973; King, 1993). In its classical form, the model of chromosome speciation was based on the statement that individuals that were heterozygous for chromosome rearrangements had reduced fitness because of meiotic irregularities resulting from structural differences in maternal and paternal karyotypes (Lorkovic, 1974, 1979; Gropp et al., 1982; John et al., 1983; Piálek et al., 2001). However, meiotic irregularities also may be due to differences in genes rather than differences in chromosomes (Dobzhansky, 1933, 1937; Coyne and Orr, 1998). In general, it is hard to separate the effects of genetic (molecular) and karyotypic (chromosomal) divergences in establishing a sufficient barrier to gene flow between species. To examine this problem, we compared genetic and karyotypic divergences using both cytogenetic and molecular approaches. According to our data, the genetic divergence among Agrodiaetus species can be small, even when differences in chromosome numbers between the same species are large. Thus the genus Agrodiaetus may present a case of species radiation that has been driven by karyotype diversification. However, this supposition requires additional investigation. For example, the role of karyotypic differences as a postzygotic reproductive barrier in Agrodiaetus needs to be assessed. Chromosome rearrangements do not always result in hybrid sterility, and so far data regarding chromosome behavior in interspecific hybrids of Agrodiaetus are lacking.
The available data from interspecific crosses suggest that structural chromosome rearrangements and meiotic irregularities in Lepidoptera do not have a simple relationship. Fertile hybrids of the silkmoths Antheraea roylei (n = 31) and A. pernyi (n = 49) demonstrate that major differences in chromosome number are compatible with regular meiotic segregation. In F1 males, 31 chromatin bodies were observed at metaphase I. These were interpreted to be 13 bivalents and 18 trivalents (Nagaraju and Jolly, 1986). The hybrids were interbred for many generations, and in F23 and F32 males 49 chromatin bodies were observed at metaphase I. Thus, there seems to have been selection against the larger chromosomes of A. roylei. Nagaraju and Jolly (1983) did not provide evidence as to whether recombination had resulted in introgression of genes from A. roylei onto A. pernyi chromosomes. By contrast with these Antheraea hybrids, hybrids among multiple morphologically distinct Papilio species with indistinguishable (n = 30) karyotypes have shown that major disturbances of synapsis, and resultant infertility, can occur in the absence of obvious structural rearrangements (Maeki and Ae, 1976, 1978a, 1978b). In fertile hybrids, 30 chromatin bodies were observed at metaphase I (interpreted as 30 bivalents formed by complete synapsis), whereas in infertile hybrids, higher numbers of chromatin bodies were observed (interpreted as a mixture of bivalents and unpaired univalents).
Because of regular segregation of trivalents and the difficulty of distinguishing trivalents from bivalents cytologically the question arises as to whether fission/fusion polymorphisms within species could go unrecognized. Most meiotic chromosomes of Lepidoptera are dotlike under standard microscopy. Diploid chromosome numbers are usually obtained by doubling the number of chromatin spots observed on metaphase plates during male meiosis I. The spots are thus assumed to be bivalents. Heterozygosity for a chromosomal fission would result in a trivalent rather than a bivalent without changing the observed number of spots, whereas fission homozygotes would have one more spot on metaphase plates. Microscopic discrimination of trivalents or higher order multivalents from bivalents may be difficult for many lepidopteran cells.
The rates of karyotype evolution we infer in Agrodiaetus appear inconsistent with every chromosomal rearrangement having been associated with a substantial heterozygote disadvantage. Therefore, the possibility that chromosomal rearrangements may not have imposed absolute barriers to gene flow (at least at early stages of karyotype divergence) should be considered as a possible explanation of apparent inconsistencies between the recovered molecular phylogeny and chromosome numbers. This view would call into question the practice of describing new Agrodiaetus "species" solely on the basis of sympatric occurrence of individuals with different chromosome numbers but similar morphology. One would need evidence that the observed karyotypic differences are stably maintained and that the local population does not contain individuals with intermediate numbers. This evidence is available in some cases. For example, A. interjectus was described by Lesse (1960a) on the basis of its profoundly different karyotype (n = 29–32; later this taxon was included into A. eriwanensis with n = 34; Lukhtanov et al., 2003) and was separated from the phenotypically similar and sympatrically distributed A. ripartii (n = 90), A. demavendi (n = 67–70), and A. alcestis (n = 20). After these species were described by Lesse, they have been routinely collected by many researchers from sympatric populations (e.g., Lukhtanov et al., 2003) Therefore, the impact of karyotype changes in preventing gene flow between these taxa and their validity as reproductively isolated species seems to be well supported. Lukhtanov (1993) presented an evolutionary model of karyotype diversification in Agrodiaetus to explain such examples of highly divergent, but stable chromosome differences within sympatric populations. In this model, chromosomal differences between closely related populations accumulate in allopatry and constitute barriers to hybridization when descendants are secondarily brought into sympatry.
Lepidopteran genera with wide ranges of chromosome numbers have also been described outside of the Lycaenidae. Haploid chromosome numbers in Leptidea (Pieridae) range from 26 to 104 (Lorkovic, 1941; Lesse, 1960b) and in Erebia (Nymphalidae) from 8 to 52 (Lesse, 1960a; Lorkovic, 1972; Lukhtanov, 1987). An extreme range, comparable to that observed in Agrodiaetus, has recently been described in scale insects of the genus Apiomorpha (Hemiptera: Eriococcidae). Haploid complements in this genus range from 2 to 96 (Cook, 2000). It is probably no coincidence that Agrodiaetus (Lepidoptera) and Apiomorpha (Hemiptera) both belong to insect orders that have holocentric chromosomes (Hughes-Schrader and Schrader, 1961; Kuznetsova, 1979; Maeki 1980a, 1980b; Wolf et al., 1997). Centromeric activity of holocentric chromosomes is not localized to a single site but is spread throughout the chromosome. As a consequence, the fragments from a chromosomal fission can attach to mitotic and meiotic spindles and be protected from loss at cell division.
Despite the above arguments for a possible role of holocentric chromosomes in karyotypic diversification, karyotypes of most Lepidoptera are highly conservative (Robinson, 1971). The existence of well-defined modal chromosome numbers, for the Lepidoptera as a whole and for the family Lycaenidae, suggests that chromosome numbers are stable in most evolutionary lineages. Either bursts of karyotype diversification, such as the one documented here for Agrodiaetus, are rare and when they occur leave few long-term descendant species, or perhaps some selective force favors a return to the modal chromosome number.
Our conclusion that karyotypic instability has originated at least three times in the section Polyommatus suggests that polyommatines may possess some factor predisposing them to chromosomal rearrangement. Transposable elements cause chromosome rearrangements in plants (Bennetzen, 2000) and animals (Lyttle and Haymer, 1992; Cáceres et al., 1999; Lönnig and Saedler, 2002) and may be horizontally transferred between species. For example, P elements are absent from all strains of Drosophila melanogaster collected before 1950 but are present in most strains collected since the 1950s (Kidwell, 1983). P elements appear to have been transferred to D. melanogaster from D. willistoni during the 20th century and have rapidly spread through the global population of D. melanogaster (Clark and Kidwell, 1997). During their initial spread into previously uninfected genomes, P elements cause a suite of genetic disturbances known as hybrid dysgenesis, including a greatly increased rate of chromosome rearrangement (Engels and Preston, 1981). In the virilis group of Drosophila, transposons have been implicated in the origin of many chromosome rearrangements, both those that are fixed differences between species and those that are polymorphic within species (Evgen'ev et al., 2000). It is tempting to speculate that karyotypic diversification in Agrodiaetus may have been driven by the spread of a transposable element, perhaps with horizontal transfer to or from the related genera Lysandra and Plebicula.
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Acknowledgment |
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We thank Andrea Sequeira and Brian O'Meara for their valuable help in using phylogenetic software programs and Zsolt Balint, Mark Cornwall, Brian Farrell, Gonzalo Giribet, James R. Morris, Swee Peck Quek, Ted Schultz, Chris Simon, Roger Vila, and two anonymous reviewers for their insightful comments on the manuscript. We are indebted to Brian Farrell for allowing us to use equipment in his lab. We are grateful to Ehab Abouheif, Antonio Barbadilla, Can Bilgin, Fidel Fernández-Rubio, Grigorii Grigorjev, Jordi Jubany, Carlos Ibánez, Mike Lockwood, Ramón Martínez, Doyle McKey, Miguel L. Munguira, Carles Palau, Sergei Sazonov, Arthur Shapiro, Constantí Stefanescu, Mark Travassos, Roger Vila, and Vera Zurilina for providing advice and/or collecting specimens. The Putnam Expedition Fund of the Museum of Comparative Zoology generously funded three collecting trips (in 2000, 2001, and 2002). Additional support for this project came from NSF DEB-0309287 (to N.P.K. and N.E.P.), the Baker Foundation (to the N.E.P. laboratory), Harvard College Research Fund (to J.W.S.C.), Harvard University (to D.H.), NSF DEB-9615760 (to N.E.P.), and the Russian Foundation for Basic Research (RFFI 02-04-49138) and the Russian Federal Programs Universities of Russia (UR 07.01.056) and Leading Scientific Schools (NSH-2232.2003.4) (to V.A.L.).
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