A classification of Danaus butterflies (Lepidoptera: Nymphalidae) based upon data from morphology and DNA Smith, David A. S. Lushai, Gugs Allen, John A. Zoological Journal of the Linnean Society 2005 2005-06-30 144 2 191 212 343SP ISSUE [390,566,772,791] Insecta Nymphalidae Danaus Animalia Lepidoptera 8 199 Arthropoda species gilippus  Tamura- Nei GDs, based upon mtDNA (12S + COI loci), between  gilippusand the  chrysippus s.l.subtaxa are as follows: dorippus-1, 6.3%;  petilia, 5.2%;  chrysippus s.s., 4.9%; these distances may be compared with a mere 0.1% that separates  gilippusfrom  eresimus. And yet, whereas  gilippusand  eresimusare reproductively isolated and morphologically distinct species that are sympatric over a huge geographical area,  gilippuslacks observed structural apomorphies compared to the allopatric and genetically distant taxa that comprise  chrysippus s.l.(A & V-W;  Smith et al., 2002for discussion). The similarity of  gilippusand  eresimushaplotypes implies that speciation has occurred only within the last 40 000 years or so ( Lushai et al., 2003b). As the two species are now sympatric over most of their combined geographical range, cladogenesis too may have been sympatric, though allopatric or parapatric scenarios are at least equally plausible ( Coyne & Orr, 2004). If the gilippus-eresimusspeciation was sympatric, prezygotic isolation through genitalic and species recognition markers (characters 36, 46, 54, Appendix 1A, B), may have been enhanced, either before speciation by reinforcement, or subsequently by reproductive character displacement ( Butlin, 1989). Moreover, if cladogenesis occurred only millennia ago (  Smith et al., 2002; Lushai et al., 2003b), the  gilippusand  eresimusclusters must have acquired prezygotic isolation within that short time.  Lushai et al. (2003b)applied molecular clocks for the COI gene in  Alphaeusprawns ( Knowlton et al., 1993) and the 12S locus in  Littorina(gastropod molluscs), Reid, Rumbak & Thomas (1996)to the  gilippus+  eresimusand  chrysippus s.l.haplotypes. The mean of the two correlated clock rates suggests that cladogenesis between these groups occurred ~2.8 million years ago (Mya), while divergence of the  gilippus+  eresimusand dorippus-1matrilines must have been even earlier, around 4.1 Mya. These calculations indicate that the divergence of  gilippus+  eresimusfrom all  chrysippus s.l.taxa occurred in the Pliocene, 3–4 Myr before the gilippus-eresimusdichotomy. Thus, the morphological features (characters 46, 54, 55, Appendix 1A, B) that distinguish the  gilippus+  chrysippus s.l.cluster from  eresimus( Fig. 2B) are probably symplesiomorphic, not synapomorphies as believed by A & V-W. It follows that butterflies with  gilippus+  chrysippus s.l.structural morphology must have dispersed to the Americas from the Old World early in the history of the genus, whereas the distinctive morphological features of  eresimus, in particular characters 46 and 54 (Appendix 1A, B), are apomorphic and of more recent Neotropical origin. An alternative scenario is that  gilippusand  eresimushad originally evolved distinct mitochondrial genomes but subsequent hybridism, possibly a rare or localized event, has resulted in the introgression of cytoplasm from one species to the other ( Lushai et al., 2003b) and thus erased the matrilineal history of the introgressed species. The possibility that such an event might be unique to Grand Cayman, where the samples of both species were collected, has to be acknowledged. However, it is unlikely that two relatively large and, moreover, highly vagile species would remain isolated on a far from remote island for long. This hypothesis could, however, be tested by sequencing mtDNA from sympatric sample pairs in other parts of their shared range.  Lushai et al. (2003a, 2005b) have produced evidence to suggest that hybridism in East Africa among partially isolated subspecies of the  D. chrysippuscomplex is catalysed by female-biased sex ratios that result from male-killer  Spiroplasmainfections ( Jiggins et al., 2000; see below). If similar events occurred in the recent history of  gilippusand  eresimus, it could account for the mutual convergence and low diversity of their haplotypes ( Hurst, Hurst & Majerus, 1997) in our samples ( N= 8 for both taxa). Although Sperling (1993)has shown that mtDNA is unlikely to cross species boundaries in  Papiliobutterflies due to the Haldane effect in heterogametic females, he did find one apparent exception in crosses between  Papilio multicaudatusand  P. rutulus.