BiohgicalJournal of tk Linnean Society (1997), 60: 297-316. With 9 figures

Phylogeny of the neotropical tribe Josiini (: Dioptinae): comparing &d Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 combining evidence from DNA sequences and morphology

JAMES S. MILLER, ANDREW V.Z. BROWER AND ROB DESALLE Department of Entomology, American Museum $Natural Histoy, Central Park West at 79th Street, .New York, Ml0024-5192, U.S.A.

Received 17 Nouember 1995, acceptedfor publication 19 April 1996

DNA sequences were gathered from mitochondrial COII and nuclear ribosomal 18s and 28s genes for 21 moth species in the tribe Josiini (Notodontidae: Dioptinae) and two outgroup genera. These data complement a previously published morphological character set for the same taxa. We examine whether relationships in the Josiini are best reflected by a single phylogenetic analysis of all the data, or by a consensus of separate trees generated from DNA and morphology. Even in cases where analyses of partitioned data produce incongruent cladograms, the underlying disagreement between partitions is relatively small. While both molecular and morphological data provide useful character information by themselves, we conclude that the best supported phylogenetic hypothesis is the one derived from combined analysis. 01997 The Linncan Society of London

ADDITIONAL KEY WORDS:-Cladogram - congruence - parsimony - ribosomal DNA - mitochondrial DNA - total evidence.

CONTENTS

Introduction ...... 297 Methods ...... 298 Study system ...n...... 298 DNA manipulations ...... 299 Data analyses ...... 299 Results ...... 300 Cladograms ...... 300 Congruence within and between data sets ...... 303 Discussion ...... 306 Molecules and morphology ...... 306 Combined versus separate analysis ...... 311 Acknowledgements ...... 314 References ...... 314

INTRODUCTION

The increasing use of DNA sequence data in phylogenetic analysis has sparked a lively discourse over the treatment of characters from multiple sources. The crux of

297 0024-4066/97/020297 + 20 $25.00/bj960 101 01997 The Linnean Society of London 298 J. S. MILLER ET AL. the debate lies in whether different types of characters, such as those from DNA and morphology, should be used to generate separate trees, with topological concordance being viewed as the most reliable estimate of phylogeny, or whether phylogeny is best reflected by a single parsimony analysis of all the data combined. Even though systematists believe that the phylogeny of most taxa is represented by a single tree, proponents of separate analysis argue that, under certain circumstances, alternate

data sets for the same organisms may imply vastly different cladograms (Bull et ul., Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 1993; de Queiroz, 1993; Miyamoto & Fitch, 1995; Huelsenbeck, Bull & Cunningham, 1996). These authors worry that combining heterogeneous data could produce an incorrect phylogeny. In contrast, the champions of combined analysis stress that, while certain characters may be better than others at revealing phylogeny, all potentially provide evidence of relationship. The most reliable estimate of phylogeny should therefore be based on a parsimonious interpretation of all the characters at hand (e.g. Miyamoto, 1985; Kluge, 1989; Barrett, Donoghue & Sober, 1991; Chippindale & Wiens, 1994; Nixon & Carpenter, 1996). Among the numerous papers surrounding this controversy, surprisingly few address the issues as they relate to actual data. Much discussion, especially that favouring separate analysis, is based on phylogeny simulation models or on hypothetical situations claimed to occur during the evolutionary process. The question arises: How applicable are these arguments to empirical examples? Here, we present new DNA sequence data for a tribe of neotropical called the Josiini (Notodontidae: Dioptinae), and examine the phylogenetic results in light of cladograms generated from comparative morphology of adult and immature stages (Miller, 1996).By performing both separate and combined analyses of the molecular and morphological data sets, we compare the character information found in each data type, and we evaluate whether combined or separate analyses are preferable. Our findings support other recent studies (e.g. Bousquet, Strauss & Li, 1992; Donoghue & Sanderson, 1992; Omland, 1994): (1) Morphology and DNA both provide useful characters for identifjring clades, although in this case morphology provides more robust support for inferred relationships. (2) There is general topological agreement between the cladograms produced by separate analyses. (3) The best-supported phylogenetic hypothesis is the one derived from simultaneous analysis of all the data combined.

METHODS

study sysm

The tribe Josiini, containing approximately 100 species, is currently placed in the subfamily Dioptinae (Miller & Otero, 1994). The Dioptinae are a derived clade within the Notodontidae (Miller, 199 1)) commonly called prominent moths. Dioptines are unique among prominents in that most are day-flying rather than nocturnal. Adult Josiini exhibit bright, presumably aposematic, colour patterns, and they participate in mimicry rings that include other dioptines as well as members of unrelated moth and butterfly families (Seitz, 1925; Hering, 1925). The larvae feed almost exclusively on Pa.ssy7oru (Passifloraceae), the foliage of which contains cyanogenic glycosides (Spencer, 1988))chemicals thought to confer protection to the herbivores that ingest them (Brower & Brower, 1964). MOLECULES AND MORPHOLOGY IN THE JOSIINI 299

We analyse relationships among 21 species of Josiini and two outgroup genera using DNA sequence data from nuclear ribosomal RNA genes and from the mitochondrial cytochrome oxidase I1 gene. We compare the results with Miller's (1996) morphological study in which 24 josiines were scored for 86 characters of the adults, larvae and pupae. Our comparisons involve only the 2 1 taxa shared between the two studies. Collecting localities for the study species, all in Central and South

America, are listed in Miller (1996: table 1). We chose the mitochondrial COII gene Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 as our primary source of DNA characters based on the published utility of this gene region in other species-level systematic studies of (Brown et al., 1994; Brower, 1994). Our data set includes two non-contiguous fragments of COII sequence. We also sequenced two of the most variable expansion segments of the linked 18s and 28s nuclear ribosomal subunits. Since these are believed to evolve slowly (Hillis & Dixon, 1991), we suspected that they might provide character support for basal nodes in the josiine cladogram.

DNA manipulations

Genomic DNA was extracted from individual caterpillars that had been field- preserved in 70% ethanol. Some larvae had been boiled in water briefly prior to immersion in alcohol in order to preserve colour patterns for morphological analysis. This procedure apparently did not affect subsequent DNA extraction or manipula- tion. For two species, DNA was extracted from adults that had been lab-reared and frozen at -70 "C. We follow two qualitatively similar extraction protocols, from Brower (1994) and from DeSalle, Williams & George (1993). Mitochondrial COII segments were amplified from genomic DNA via PCR using Brower's (1994) protocol with primers S3126, S3291 and A3772. Amplification of ribosomal segments employed primers 18Sa 0.7 and 18Sb 3.0 (W. Wheeler, pers. comm.) and 28s D3A and D3B (Nunn et al., 1996). Double-stranded PCR products were sequenced following the method of Thomas & Kocher (1993), and visualized by autoradiography. DNA voucher material is deposited at the American Museum of Natural History. Each gene region was sequenced in two noncontiguous blocks: 313 bases of 28s and 202 bases of 18s comprise the rDNA, while the mtDNA consists of two segments of COII corresponding to positions 3160-3442 and 3563-3742 in the Drosophila yakuba genome (Clary & Wolstenholme, 1985). The two blocks of data for each region were united to form a mitochondrial and a ribosomal data set. We hypothesized that each region might evolve as a single allele due to concerted evolution in rDNA (reviewed by Hillis & Dixon, 199 l), and to lack of recombination in mtDNA (reviewed in Harrison, 1989).

Data anahses

Sequences were aligned by eye because there was no ambiguity caused by gaps; few gaps were inferred in the RNA regions (two autapomorphies in the aligned 18s sequence) and these were short, while gaps were absent in the mtDNA fragments. Aligned sequences are available on the AMNH web page: (HTTP:/ /research.amnh.org/molecular). 300 J. S. MILLER ET AL. The search for most parsimonious cladograms was performed using PAUP version 3.1.1 (Swofford, 1993). Multistate morphological characters were treated as either additive or nonadditive according to the previous analysis (see Miller, 1996), while all DNA characters were treated as nonadditive. Ambiguous sites in the sequence data were coded as missing. Differential character weighting was not applied a prior;. Approximate searches were conducted by tree bisection-reconnection branch-

swapping, with at least ten random-addition sequence iterations per search. We use Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 strict consensus trees to summarize multiple equally parismonious cladograms for a given data set. To choose among equally parsimonious trees, we reanalysed the data using successive approximations weighting (SAW; Farris, 1969), as implemented in PAUP. Weights were assigned based on the maximum value of the rescaled consistency index among all trees, and rounded to the nearest integer, on a base weight of 1000. All trees were rooted with ,@umtha; Zthrautes and Zunacetha 2 were included to test the monophyly of the Josiini. For the cladograms produced from morphology (Fig. 1) and the combined data set (Fig. 7), we inferred branch support (Bremer, 1988, 1994) by running successive searches that retained all trees longer than the previous search, recording the number of additional steps at which particular branches collapse. This procedure was terminated in cases where search times became prohibitively long due to excessive numbers of trees saved (i,e. more than eight extra steps). We present branch support values only as an indication of branch robustness. Numerical values are not strictly comparable between cladograms or between nodes on a single cladogram; they are not meant to imply statistical confidence. The homoplasy contributed by each component data set to the combined cladogram (Fig. 7) and incongruence between data sets were measured by (D), the incongruence length difference (Mickevich & Farris, 1981; Farris et al., 1994). The significance of these values was assessed using the ARN jack-knife resampling program (Farris et al., 1994). These results are reported in Table 1.

RESULTS

Separate analyses are useful in understanding differences among data sets (de Queiroz, Donoghue & Kim, 1995; Nixon & Carpenter, 1996). In addition to a basic division between morphology and DNA, we chose to further partition the morphological characters into those from adult vs. immature stages, and we divided the molecular data into two sets of linked sequences, mitochondrial and ribosomal. For the 23 taxa used in our study, analysis of morphology alone yielded two equally parsimonious ( = EP) trees. The consensus (Fig. 1) differs from Miller’s (1 996) completely dichotomous cladogram only in that relationships among tur-a,3. augua and 3. aurifsa are unresolved. Reanalysis after SAW gave one fully-resolved cladogram with the group u. turgula + v.aunhu + J. aursflua]]. When the 59 adult characters were analysed separately from those of immature stages, only one additional node collapsed [iThirmida superba + T. dkcinota + Cyanotricha necyria] in a consensus of eight EP trees. The 27 larval and pupal characters provided much less resolution, yielding 371 EP trees. Possible explanations for the difference between MOLECULES AND MORPHOLOGY IN THE JOSIINI 30 1 adult and immature data are discussed in Miller (1996). Results from the ARN test showed no significant difference between the two morphological partitions. The mtDNA data yielded 29 EP trees, with their consensus showing lack of resolution among basal clades (Fig. 2). Reanalysis using SAW gave one of these original 29 trees (Fig. 3), with the clade [C. necyria + [T.superbu + [T. dzihnotu + J. mu]]] as sister to the Jluoniu-jluuissimu clade. The hypothesis of josiine relationships

implied by mtDNA is quite different from the one supported by morphology in Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 several important respects. For example, Figure 3 shows the outgroup genus Tithraustes nested within the Josiini as sister taxon to [w.radium + J. ligata] + 3. g@nteu]. Furthermore, the phylogenetic positions of J. megaera and J. druconzi differ drastically between the morphology and mtDNA trees. There are other discrepancies. Parsimony analysis of the ribosomal sequence data produced 830 EP trees. The consensus (Fig. 4) identified few clades in the Josiini (only 7 of 21 possible nodes). Among those that do appear, the clade comprising [Tithruustes + J. uuntu] + J. fornax] is untenable (see discussion). Several rounds of SAW reached an asymptote of 10 equally parsimonious trees, the consensus of which provided greater resolution

- J. turgida 2 J. aurlflua - J. aurifusa 2

5

J. insincera ,- ,- J. fiuonia J. annulata J. cruciata J. striata J. gopala J. fornax

G. baetifica J. draconis T. superba C. necyria T. discinota J. rnegaera Tithraustes Zunacetha Zunacetha 2 Figure 1. Strict consensus of two equally parsimonious (EP)cladograms for the morphological data found in ten heuristic search iterations. For this and all following trees, the consistency index (CI) is calculated after excluding uninformative characters. Length = 200 steps, CI = 0.644, RI = 0.849. Estimated branch support values (Bremer, 1994) are shown above each resolved node. 302 J. S. MILLER ET AL. (an additional eight nodes; tree not shown), but little congruence with trees from the other data partitions. These results suggest that ribosomal genes are not particularly informative on basal splits within the josiine phylogeny. A combined matrix of DNA sequence data produced 3 1 EP trees. The consensus of those trees (Fig. 5) is no more resolved than either of the consensus trees from the mtDNA or rDNA alone (Figs 2 and 4). However, some clades supported by

morphology were recovered, such as the groups containingJosia lkatu andJ. aun&a. Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 In addition, DNA characters corroborated sister species relationships, such as 3. cruciatu + 3. annulutu and J. stria& + 3. gopala, that are supported by numerous morphological synapomorphies (Miller, 1996). Analysis of the 31 DNA trees using SAW gave a single fully-resolved cladogram. That tree (Fig. 6) differs from any of the original DNA cladograms produced by equal-weighting. It shows important similarities with the morphological tree (Fig. l), as well as clear differences. For example, the positions of Zthrawtes andJosia megaera (Fig. 6) are in stark conflict with their placement on the morphological cladogram. When all available molecular and morphological data for the 23 study species were analysed in a combined matrix without character weighting, a fully-resolved cladogram resulted (Fig. 7). Not surprisingly, SAW gave this same tree. Figure 7

J. turgida J. aurlfusa J. auriflua J. fluonia J. fornax J. annulata J. cruciata J. striate J. gopala J. draconls H-1 J. flavissima J. radians I J. ligata J. gfgantea T. superba C. necyria J. insincera Tithraustes G. baetlfica T. discinota J. ana J. megaera Zunacetha &Zunacetha 2 Figure 2. Strict consensus of 29 EP trees for the mitochondrial sequence data found in ten heuristic search iterations. Length = 381 steps, CI = 0.455, RI = 0.487. MOIJXULES AND MORPHOLOGY IN THE JOSIINI 303 differs from the morphology tree (Fig. 1) in only two ways. First, rather than showing an unresolved trichotomy for the clade containingJ. turgida, J. aunfia andJ. aurgflua, the combined analysis indicates that the first two are more closely related than either is to aurgfua. The second way that the morphology and combined analysis trees differ is in the position of Josia fomax. According to the hypothesis based on all data, J. fornax andJ.Juonia are sister taxa (Fig. 7), whereas the inference from morphology is

thatJ.fomax is basal to a clade containingJ.Juonia and four other species (Fig. 1). We Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 accept the cladogram in Fig. 7 as the best one available until new evidence, such as the addition of taxa or novel characters, is brought to bear.

Congruence within and between data sets

Table 1 summarizes numerical attributes of the trees discussed above. Most important are the values indicating levels of homoplasy intrinsic to the various data subsets, and the extra homoplasy introduced due to incongruence between data sets when they are combined (D; Mickevich & Farris, 1981). The homoplasy inherent within partitions is approximately an order of magnitude larger than the homoplasy

- J. striata - J. gopala J. draconis - J. flavissima T. superba T. discinota -

Zunacetha Zunacetha 2 Figure 3. Single tree derived from Successive Approximations character weighting (SAW) applied to the 29 trees from equal-weighted analysis of the mtDNA data. This tree has the same topology as tree 28 from that search. 304 J. S. MILLER ET AL. between data sets (Table 1). Thus, even though tree topologies inferred by analysis of data subsets differ from one another, and the ARN test shows significant heterogeneity between the morphological and molecular partitions, the characters contradict one another to a much greater degree within than between partitions. Authors who have expressed concern about combining data sets that show heterogeneity (Bull et aL, 1993; De Queiroz, Donoghue & Kim, 1995; Huelsenbeck,

Bull & Cunningham, 1996) offer no easy means to infer relationships under such Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 circumstances. Our results suggest that moderate heterogeneity among partitions does not have a negative effect on simultaneous analysis. Another aspect of combining morphological and molecular data is shown in Table 2. Here, estimated branch support values (Bremer, 1994) are compared at each cladogram node for the trees from morphology and from all data. Support for individual branches is increased at 13 nodes by addition of molecular characters, while it is reduced at only four nodes. Total support (sensu Kdlersjo et al., 1992) across the entire topology is increased by 28 steps (37%) through addition of the molecular data. Increased branch support is not evenly distributed throughout the cladogram, however, so this may not be a relevant measure of robustness for the tree as a whole (Bremer, 1994).

J. turgida J. radians J. auriflua J. gigantea J. iigata J. insincera J. fluonia J. annulata J. cruciata J. striata T. superba C. necyria T. discinota J. aurifusa I Tithraustes J. fornax J. flavlssima G. baetifica J. gopaia J. ena J. megaera J. draconis Zunacetha Zunacetha 2 Figure 4. Strict consensus of 830 EP trees for the 28s and 18s rDNA sequence data, found in ten heuristic search iterations. hngth = 147, CI = 0.627, RI = 0.670. MOLECULES AND MORPHOLOGY IN THE JOSIINI 305 Advocates of topological congruence (e.g. Lanyon, 1993; Miyamoto & Fitch, 1995) suggest that the most reliable phylogeny is reflected by a consensus of fundamental trees from different data sets. Figure 8 shows a consensus of the trees from morphology (Fig. 1) and DNA (Fig. 5). This cladogram is indeed highly conservative; the onlyjosiine clades retained are species groups, such as the ligata and aunia groups, or species pairs, such as J. annulata + J. cruciata. However, in comparison with the tree produced by simultaneous analysis (Fig. 7), a great deal of Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 information about the phylogenetic history of the Josiini has been lost. Not only does the tree in Figure 8 obscure hypotheses about interrelationships among major josiine groups, but some groups that are strongly supported by one or several partitions disappear due to lack of support from others. For example, both morphology (Fig. 1) and rDNA sequence data (Fig. 4) support a clade comprising the high Andean species ?hirmida superba, irhirmida discinota and Cyanotricha necyria, long regarded by taxonomists as being close relatives (Prout, 19 18; Hering, 1925). However, this clade disappears in a consensus of morphological and molecular trees because it was not supported by characters from mtDNA (Fig. 2).

J. turgida J. aurifusa J. auriflua J. fluonia

IJ. radians PJ. ligata

J. cruciata J. striata

~ J. gopala - T. superba C. necyria J. flavissima J. insincera J. fornax Tithraustes G. baetifica T. discinota 306 J. S. MILLER ET AL.

DISCUSSION

Molecules and morphology

More than a decade has passed since the hopell days when DNA data were being touted as the ultimate answer for elucidating organismic phylogeny (e.g. Selander,

1982; Gould, 1985). Rather than being free from the difficulties inherent in Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 morphological characters, such as homology determination and character coding, it has become clear that DNA data fall prey to the same problems for systematic inference (Patterson, Williams & Humphries, 1993). One of the supposed strengths of molecular data was 'the vast amount of character information that would potentially be made available. In fact, concern was raised that data from molecules would swamp the signal from morphology (Kluge, 1983). Such fears appear unwarranted: while a molecular data set may contain thousands of aligned nucleotide positions, the number of cladistically informative DNA characters rarely overwhelms a robust morphological data set (Miyamoto, 1985; Patterson, Williams & Humphries, 1993; Chavarria h Carpenter, 1994). For the Josiini, although we

J. turgida J. aurifusa

J. llgata J. gigantea J. insincera J. megaera

- J. annulata - J. cruciata - J. striata J. gopala - J. flavissima

Tithraustes T. dlscinota

1 Ii:irz:ca Zunacetha Zunacetha 2 Figure 6. Single tree derived from SAW applied to the 3 1 trees from equal-weighted analysis of all DNA data. This tree topology does not correspond to any of the original trees. MOLECULES AND MORPHOLOGY IN THE JOSIINI 307 sequenced nearly 1000 bases per individual, there were 137 informative characters from DNA compared to 80 from morphology (Table 1). An emerging reconciliation in the debate concerning the virtues of molecules versus morphology is that each type of data offers advantages and disadvantages, and neither is inherently superior (Hillis, 1987; Omland, 1994; Chippindale & Wiens, 1994).The judicious combination of both may provide insights that were not evident

from prior analysis of less inclusive data sets. This statement appears to hold true for Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 organisms spanning the taxonomic spectrum. For example, it has been shown for plants (Donoghue & Sanderson, 1992; Smith & Sytsma, 1994), moths (Brown et aL, 1994), bees (Chavarria & Carpenter, 1994), sea urchins (Littlewood & Smith, 1995), ducks (Omland, 1994) and primates (Yoder, 1994). There are clearly differences in the cost of molecular versus morphological research, and given the current climate of limited funding for systematics, the latter may be preferable as a first line of attack. Our study, while ultimately gaining support from both sources of data, provides an example in which morphological characters appear, by several measures, to provide

J. turgida J. aurifusa

J. radians J. ligata J. gigantea I LJ.insincere J. fluonia

J. annulata J. cruciata J. striata J. gopala

G. baetifica J. draconis T. superba C. necyria T. discinota

Tithraustes Zunacetha Zunacetha 2 Figure 7. The single most parsimonious tree (found in 100 search iterations)resulting from analysis of all data combined. Length = 765 steps, CI = 0.505, RI = 0.650. Minimum and maximum branch lengths (number of steps) under alternate character optimizations, as calculated by McClade (Maddison & Maddison, 1992), are indicated by the shaded bars. Acctrans branch lengths as reported by PAUP are indicated above branches, and estimated branch support values are indicated below internal branches. W 002

TABLE1. Structure within and among different data partitions. Numbers of variable and informative characters are indicated. Minimum length represents the lowest possible number of state changes per character, summed for all characters. Intrinsic homoplasy is the minimum additional number of steps required to hierarchically arrange the data beyond the miniium length. D homoplasy is the extra homoplasy contributed by combining data in simultaneous analysis.ARN scores represent the proportion of jack-knifed random partitions resulting in trees as short or shorter than the observed trees, based on 999 samples 5 Associated Number of Minimum Number Shortest tree Intrinsic D ARN v, Character source figure characters length of trees discovered homoplasy homoplasy scores

Adult morphology t 59v/54i 97 8 146 49 (34%) - - E Larval/pupd morphology t 27v/26i 34 371 52 18 (35%) - - P 2 morphology 1 86v/8Oi 131 2 200 67 (34%) 2 (1%) 0.189 3 COII 2 163v/99i 208 29 381 173 (45%) - - 18 Sand 28 S 4 97v/38i 116 830 147 31 (21%) - - $ I: molecules 5 26Ov/l37i 324 31 551 204 (37%) 23 (4%) 1.000 2 all data 7 346v/2 17i 455 1 765 271 (35%) 14 (2%) 0.025

+See Miller (1996, Fig. 22). Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 September 27 on guest by https://academic.oup.com/biolinnean/article/60/2/297/2705884 from Downloaded MOLECULES AND MORPHOLOGY IN THE JOSIINI 309 more robust evidence of relationships than those from DNA (see also Chavarria & Carpenter, 1994). Using simple resolving power of data partitions as a measure of success, DNA characters do not fare well compared to morphology. While morphology produced only two trees (Fig. l), analysis of molecular data resulted in 31 equally parsimonious trees, the consensus of which is poorly resolved (Fig. 5). Partitioning the DNA data into ribosomal and mitochondrial sets shows that both

types of characters have problems deciphering relationships in the Josiini. The Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 mitochondrial characters produced 29 EP trees (Fig. 2), and the ribosomal data produced 830 EP trees (Fig. 4). Neither result is well resolved, and both contain groups that are incongruent with the topology derived from all characters. Successive approximations weighting of the DNA trees produced a somewhat more satisfying result. The SAW tree (Fig. 6) reflects much of the same structure apparent from the combined analysis (Fig. 7): 11 of 21 clades are congruent, and most of the disagreement is due to the differing positions of taxa with many autapomorphies. Although rarely applied to DNA studies, this finding suggests that SAW may have considerable potential in molecular systematics. Nevertheless, as noted in the results, the most parsimonious cladogram after SAW is still substantially incongruent with the cladogram from simultaneous analysis. A disadvantage of SAW is that the differential weighting makes comparisons of relative branch support and estimation of incongruence length differences among partitions impossible. This is especially problematical in cases like ours, where a SAW tree has topology different from any of the fundamental trees that were used to assign weights (Figs 5, 6).

TABLE2. Branch support for the most parsimonious tree from all data (Fig. 7), morphology alone (Fig. l),and the difference between the two, showing the positive contribution of the molecular data to branch support for most nodes

Node All data Morphology Difference

1. turgzda+aun&sa 0 +4 2. I+aunjluu 2 +2 3. radians+ligata 3 +5 4. Stgigantea 5 +3 5. 2+4 2 t2 6. 5+insincera 5 +2 7. jluoniaifmax t +2 8. annulata+mciata 4 +4 9. 7+8 t +1 10. striata+gopala 6 +2 11. 9t10 2 +2 12. jlavissima+ena 4 +2 13. 11+12 7 0 14. G. baet@ca+draconis 3 -2 15. 13+14 3 -1 16. C. necyria+T discinota 3 -1 17. 16+ I: superba a(+) 0 18. 15t17 6 0 19. 6+18 2 -1 20. 19+megwu 8(+) 0 21. 20+Tithraustes 2 +2

*Branch support above 8 steps was not calculated, due to excessive numbers of trees . Thus, these values represent estimated minima, and actual values (and difference scores) could be higher. Nodes for which branch support was 8+ in both analyses are not directly comparable, and branch support contributed by the molecular data could be either higher or lower. tNodes that did not appear in the morphology tree. All branch support for these nodes is drawn from the molecular data, and the values in the difference column are estimated minima. 310 J. S. MILLER ET AL. Comparison of resolved nodes among trees produced by equal-weighted analyses is a more fruitful approach for assessing topological concordance. Cladograms from DNA imply certain relationships among the Josiini that are implausible on morphological grounds. For example, the genus Ethramtes is nested within the Josiini according to both the DNA data partitions (Figs 2, 3, 5, 6). However, morphology strongly supports monophyly of the Josiini to the exclusion of

lithramtes. lithramtes, Zunacetha and nine other dioptine genera form a clade Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 supported by numerous morphological synapomorphies Wer, unpublished data), including the presence of a unique organ on the male forewing (Miller, 1989),known to produce sound (Forbes, 1922). This clade is the likely josiine sister group (Miller, unpublished data). Furthermore, lithramtes does not exhibit any morphological synapomorphies of the Josiini. Nine are now known (table 4 in Miller, 1996). It is important to note that while the DNA data imply an anomalous placement for lithraurtes in partitioned analyses, not only does lithramtes arise in the orthodox position in the combined analysis (Fig. 7), but inclusion of molecular characters increases branch support for its placement by two steps over the support from morphology alone (Table 2). Josia mgwa differs substantially in its placement by alternate data partitions.

J. turgida J. aurifiua J. aurifusa J. fluonia J. radians J. ligata J. gigantea IJ. annulata J. cruciata J. striata J. gopala I T. superba C. necyria J. flavissima J. insincera J. fornax Tithraustes G. baetifica T. discinota J. ena J. megaera J. draconis MOLECULES AND MORPHOLOGY IN THE JOSIINI 31 1 While J. megaera undoubtedly belongs within the Josiini (Figs 1, 7), it also exhibits morphological traits supporting its position as the basal species in the tribe. For example, almost all prominent moths possess a hearing organ, or tympanum, on the third thoracic segment (Miller, 1991). Dioptinae are no exception, but the tympanum of Josiini is modified to form a huge internal pouch shaped like a kettle drum (Richards, 1932; Sick, 1940; Kiriakoff, 1950). In outgroup species the

tympanal depression is smaller. The hearing organ of Josia megaera more closely Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 resembles the outgroup configuration (Miller, 1996). As another line of evidence, caterpillars of all josiines except J. megamu have a white head variously patterned with black. In other Dioptinae and inJ. megama, the head is uniformly brownish orange (Miller, 1996). The special status of this species is not apparent from the molecular data; in the SAW mtDNA tree, 3. mgma appears in a clade with the J. aur3flua group (Fig. 3). Again, however, morphological orthodoxy is retained in the cladogram derived from combined analysis (Fig. 7), and branch support for J. megama’s placement is not reduced by addition of the molecular data (Table 2). It has increasingly been recognized that DNA sequences from various gene regions are not equally effective as character systems (Friedlander, Regier & Mitter, 1994; Brower & DeSalle, 1994). The same obtains for subsets from morphology. Adult characters work best at unraveling relatively recent radiations, whereas larval characters tend to provide resolution at higher hierarchical levels (Miller, 199 1, 1996). In the Josiini, mitochondrial data did not identify the clade comprising 77zirmida and Cyanotricha, but ribosomal and morphological data did. On the other hand, mitochondrial characters may be highly efficient at resolving relationships within sibling species groups. Josia turgula and J. aunta endemic to Venezuela, and J. aur3flua from the eastern slope of the Andes in Ecuador and Peru (Hering, 1925; Bryk, 1930), are so closely related that species boundaries based on morphology are obscure (Miller & Otero, 1994). The mtDNA data produced resolution at this node (Fig. 2), and added resolution to the combined analysis (Fig. 7). Branch support values (Table 2) suggest that the molecular data are making the greatest contribution . among closely-related taxa, while offering less (or even detracting) at deeper nodes and for taxa, such as Josia draconis and baetajica, with numerous autapomorphies. In summary, perhaps the wisest position to take is that outlined by Omland (1994); data from different sources are most appropriately applied to solving different types of phylogenetic problems. Morphological systematics has a long and illustrious history, and our understanding of its character systems is fairly well established. By contrast, our exploration of DNA characters and their potential is in its infancy (Brower & DeSalle, 1994). The incorporation of morphological data into phylogenetic hypotheses is thus critical. How should this be accomplished?

Combined versus separate anabsis

If we don’t already know the ‘true’ phylogeny of a group of organisms, then the best estimate of relationships would seem to be the one that offers the simplest explanation of all the relevant data. Trying to incorporate information about grouping implied by data partitions begins a journey down the slippery slope of ad hoc justification from parsimony to a convoluted labyrinth of alternate ways to interpret character congruence. A single method is not only desirable, but necessary, 312 J. S. MILLER ET AL. to avoid this chaos. We maintain, as has been enumerated many times in the distant and recent past (e.g. see Popper, 1959; Farris, 1983; Kluge, 1989; Swofford, 1991; Panchen, 1992; Eernisse & Kluge, 1993; Wenzel & Carpenter, 1994; Brower, DeSalle & Vogler, 1996; Nixon & Carpenter, 1996), that cladistic analysis using parsimony is the only viable way to explain patterns of character state distribution. In understanding relationships within the Josiini, our method of choice is to combine

all the data and find the shortest cladogram. The tree in Figure 7 represents the best Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 estimate of evolutionary history currently available for the 21 josiine species in our study. In order to investigate particular historical ecological questions, such as the evolution of hostplant associations in these moths, a phylogenetic hypothesis is needed (Mitter & Brooks, 1983; Wanntorp et aL, 1990; Miller & Wenzel, 1995). The cladogram based on combined analysis of morphological and DNA data is the one we would use. However, there seems to be great interest among a few systematists in providing a measure of how ‘reliable’ a particular cladogram is (Lanyon, 1993; Miyamoto & Fitch, 1995; de Queiroz, Donoghue & Kim, 1995; Huelsenbeck, Bull & Cunningham, 1996). These authors voice their desire for finding the ‘true’ phylogeny, a stable solution against which evolutionary scenarios can be tested. This highlights a basic difference in views. While we agree that there actually is a ‘true’ phylogeny, we consider it an unattainable, metaphysical goal. The search for truth is an asymptotic process, approximated by an ongoing quest for the most parsimonious explanation of all relevant data. We are comfortable with the notion that every cladogram will remain an hypothesis subject to change and modification. If an ecologist provides comparative evidence for a group of organisms and the data can be converted reasonably into discrete character information, then those findings should be reflected in a revised hypothesis of relationships. To us, careful data collection, careful analysis, and maximum parsimony lead to well-supported hypotheses. Corroboration (i.e. stronger support) is implied when data from novel sources are added to an existing cladogram and the original groupings are retained. The most common argument in favour of separate analysis suggests that, because areas of agreement among trees produced by analysis of separate data partitions are especially likely to be true (de Queiroz, Donoghue & Kim, 1995), a consensus tree summarizing these would provide a highly conservative estimate of phylogeny. Our application of the procedure produced results of limited value. The josiine data can be partitioned in a multitude of ways. Some authors have noted that the rationale for choosing subdivisions is largely arbitrary (Cracraft & Helm-Bychowski, 1991; Kluge & Wolf, 1993). At the extreme, a strict consensus of all characters taken individually will always produce a completely unresolved tree, which is not particularly helpful. Any less drastic partitioning of available evidence implies relative nonindependence of the characters within each subset, which is always theoretically undesirable for either cladistic or statistically-oriented methods. Miyamoto & Fitch (1995) suggest that criteria for partitioning data can come from use of different character discovery tools and techniques (for example, a microscope versus a DNA sequencing rig). Different genes and life stages form discernibly separate character groupings, each of which potentially provides characters that corroborate an underlying phylogenetic pattern (Hennig, 1966; Mickevich & Johnson, 1976; Miyamoto & Fitch, 1995). We might therefore divide our data into MOLECULES AND MORPHOLOGY IN THE JOSIINI 313 four subsets: adult morphology, immature morphology, mtDNA and rDNA. Four consensus trees result from these four analyses. The combined consensus of those yields what is arguably the most conservative estimate of josiine relationships, one that makes no statement about hierarchical pattern (Fig. 9). This approach is obviously counterproductive; robust character support in some partitions allows us to draw inferences about relationships among species of Josiini, but these inferences are cancelled out by the absence of information in alternate partitions. The goal of Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 systematics is a cladogram that reflects accumulated knowledge, not one that completely obscures it. Analysis of the combined josiine data therefore offers both better resolution, and the philosophical satisfaction of not compromising the integrity of the analytical method by assuming independence or nonindependence of alternate data partitions. Figure 9 was produced by strict consensus of all the cladograms resulting from separate analyses of the four data partitions. Lanyon (1993) has argued that this approach is “unduly pessimistic”. As an alternative, he suggested presenting summary cladograms, termed phylogenetic frameworks, that reflect all well- supported areas of agreement between cladograms from Werent sources, even if a resolved node appears in only one of the original trees. For the Josiini, the

J. turgida J. fluonia J. radians J. annulata J. striata T. superba J. auriflua J. gigantea J. ligata J. aurifusa C. necyria - - J. flavissima J. insincera J. fornax J. cruciata Tithraustes G. baetifica J. gopala T. discinota J. ena J. rnegaera - J. draconis 314 J. S. MILLER ET AL phylogenetic framework produced by combination of morphology and DNA trees (Figs 1, 5) would be identical to the cladogram for all data analysed simultaneously (Fig. 7). We prefer to by-pass the exercise of producing a ‘phylogenetic framework‘, since it adds nothing to our knowledge and is conceptually removed from our goal of discovering the most parsimonious explanation for all available characters. We have argued against separate analysis as a general method for inferring

phylogenies, but have shown that partitioning can provide highly informative Downloaded from https://academic.oup.com/biolinnean/article/60/2/297/2705884 by guest on 27 September 2021 comparisons regarding the efficacy of different data subsets. We see little utility in treating subsets of data as nonindependent classes after they have already been incorporated into the analysis. Our josiine data show that even relatively uninformative character subsets, which produce poorly-resolved or incongruent topologies on their own, can contribute to the robustness of the combined result under cladistic parsimony. Specious models of molecular evolution based on arbitrary data partitions will not provide a substitute for the wealth of taxonomic knowledge, accumulated during two centuries of morphological systematics. We look forward to the time when character analysis of molecular and morphological data can be carried out with equal rigour.

ACKNOWLEDGEMENTS

For help with DNA data collection and analysis, we thank Elizabeth Bonwich, Ferdinand Vital and Jessica Zucker. We are grateful to Ward Wheeler and his coworkers at the AMNH for the use of 18s rDNA primers. Collection ofjosiine larvae and adults would have been impossible without the expert field assistance of Cal Snyder and Daniel Otero. Norm Platnick,Jim Carpenter, Paul Goldstein, Brian Farrell, an anonymous reviewer and the members of the AMNH molecular lab systematics discussion group provided helpful comments on the manuscript. Finally, we gratefully acknowledge the following National Science Foundation grants: BSR- 9106517 to JSM; DEB-9303251 to AVZB; BSR-9220317 to JSM and RD.

REFERENCES

Barrett MyDonoghue w,Sober E. 1991. Against consensus. syslemtic