Morphological characters alone showed little resolution in the Polistini at the species level, instead supporting most of the subgenera as monophyletic – but not all (Fig. 1). New World species as a whole were monophyletic with a bootstrap support of 75% while Old World species were paraphyletic. Of the subgenera, Polistes sensu stricto, Megapolistes, Polistella, Epicnemius and Fuscopolistes were all supported as monophyletic with bootstraps of 64%, 95%, 74%, 63% and 61% respectively. But there was no resolution within these groups, and Aphanilopterus was not resolved as a group.

The COI, and microsatellite combined characters supported the subgenera Megapolistes, Fuscopolistes, and Aphanilopterus with bootstraps of 93%, 93%, and 70% respectively (Figure 2). Both Polistella and Polistes sensu stricto were mostly supported at 50% and 66%, but each left out one species. There was also considerable resolution within the supported subgenera. Thus, the independent molecular and morphological trees offered resolution in both different and overlapping parts of the tree and, where they were resolved, were not in conflict.

The overall combined tree topology (Figure 3) is much more resolved than what was observed with the independent-character phylogenies alone (Figs. 1 and 2). The New World clade is monophyletic with 62% bootstrap support. Although the support for this same clade was 75% in the morphological characters tree (Fig. 1), the combined tree provides a much more resolved phylogeny at the species level. The clade for the Old World Polistes is paraphyletic. All the subgenera for which we have 2 or more exemplars are supported. The Old World subgenera, Megapolistes, Polistella, and Polistes sensu stricto are supported with bootstraps of 99%, 93% and 92% respectively. The New World subgenera, Fuscopolistes, Aphanilopterus and Epicnemius are supported with bootstraps of 99%, 96% and 70% respectively.

Morphological characters alone supported the Old World Ropalidiini and the New World Epiponini and Mischocyttarini with bootstraps of 64%, 85% and 91% respectively (Figure 4). All of the genera for which we had more than one species were also supported except for Polybia. There was little resolution within genera except for Brachygastra. However most genera were only represented by a few species.

The combined COI and microsatellite characters were largely uninformative on their own, though there was some grouping of epiponine species and resolution within the monophyletic Mischocyttarus (Figure 5).

The combined tree is much more resolved than either separate analysis, and bootstrap supports are greater (Figure 6). Monophyly of Ropalidiini, Mischocyttarini, and Epiponini is supported at bootstrap levels of 83%, 95%, and 90% respectively. However the relationships between the tribes are not resolved. All genera for which we have more than one species are supported except for Polybia which is broken into three groups, among which relationships are not resolved (Figure 6). Relationships among genera within Ropalidiini and Epiponini are generally not well resolved. The basal genus of the Epiponini is unresolved.

The combined character phylogeny is partly congruent with the earlier phylogeny proposed by Carpenter in 1996, though there are some differences (Figure 7). First, note that the earlier tree is not bootstrapped; Figure 1 is the bootstrapped tree for morphological characters, based on the present sample of species. Results from both studies on the monophyletic origin of the New and Old World Polistini subgenera only found support for the monophyletic origin of the New World Polistini (Fig. 3). In both studies, the Old World Polistini subgenera represent a paraphyletic group. However, the present study showed Polistes sensu stricto as the most basal subgenus (with a bootstrap support of 98%), in contrast to Carpenter 53, where it was the sister group of the New World subgenera (with a bootstrap support of 89%, Fig. 7). The present study found the sister group of the New World subgenera to be the clade of Polistella, Nygmopolistes, and Megapolistes. Several subgenera were absent from the present study (viz. Gyrostoma, Stenopolistes and Sulcopolistes) and it is possible that their absence affected character polarizations obtained within Polistes.

In contrast to Carpenter's 53 study, the present results support monophyly of the subgenus Aphanilopterus in the strict sense, and of the subgenus Epicnemius, albeit based on a smaller number of species.

Polistes sensu stricto is unique in the Polistinae for having social parasites, species that kill their host queen so her workers can rear the brood of the social parasite 12808182. The three social parasite species are monophyletic and most closely related to P. nimphus and P. dominulus, and less close to P. gallicus and P. biglumis 1282. The present study agrees that the latter two species are sister species, less close to P. nimphus and P. dominulus, but this study does not put these two as sister species. The earlier study did not include morphological characters, and did not include either P. chinensis or P. marginalis which could account for the differences 82.

The combined character phylogeny is largely congruent with the earlier phylogeny proposed by Carpenter in 1991 (Figure 8). Depending on the tribe, one or the other is more resolved, but not in major disagreement. Within the tribe Epiponini, of the polytomies observed in the Carpenter's 1991 topology, one was resolved: Agelaia and Angiopolybia are sister-groups (Fig. 8). The genus Polybia in the present study does not disagree with Carpenter et al.'s 67 conclusion that it was paraphyletic: in the present study it is unresolved, with P. (Formicicola) rejecta, P. (Trichinothorax) affinis and P. (Pedothoeca) emaciata separated from the main P. sensu stricto clade for the genus. Note that the present study is based on a broader sample of genera than in 67.

In a series of papers Strassmann and Queller argued that a form of worker control of sex ratios was general for the Epiponini 4083. This form of worker control meant that colonies did not produce a new generation of queens until the colony had reduced in queen number to one or perhaps no queens. This is important to sociality because such a queen reduction before requeening maintains high relatedness within colonies. The species that they studied this cyclical oligogyny in were Parachartergus colobopterus, Protopolybia exigua, Brachygastra mellifica, Polybia emaciata and Polybia occidentalis 6383848586. In addition West- Eberhard documented either cyclical oligogyny or some of its features in three additional genera, Agelaia, Synoeca and Metapolybia 878889. Taken together, these studies come from all of the four basal-most lineages of the Epiponini, and also cover 4 of the 6 lineages in our best sampled clade, the one containing Polybia (Figure 7). Thus, it seems they are justified in claiming the generality of this form of worker control for the tribe.

The inclusion of several genetic markers (mitochondrial and nuclear), improved the resolution of the trees obtained from morphology alone. The COI sequence alone did not resolve Polistes as a group; combining it with the microsatellites did. While COI and the microsatellite flanking regions did not resolve Polistella, adding the repeat regions did so. The use of microsatellite flanking regions and the presence/absence coding system for the repeated regions of the microsatellites proved to be useful in the resolution of some of the branches.

For the last fifteen years, a considerable rise in the use of molecular markers has been observed. The majority of the studies have exclusively included molecular data from mtDNA genes. Even when the information gathered for those studies has been informative, problems with the use of this type of molecular data (saturation synonymous sites with mtDNA; constrains imposed on RNA sites by secondary structure, length variation, etc.) have been considered 2590. Unfortunately, few nuclear markers have been used in phylogenetic systematic studies in the past. The search for new, powerful, informative-single copy nuclear markers has increased our wealth of information in systematic studies. At the present time just a few more nuclear markers have been used for insect phylogenetic studies 2691. More genes are needed for testing phylogenetic hypotheses. Meanwhile, the use of microsatellite flanking regions represents a valuable tool as nuclear single-copy markers. The wealth of microsatellite data is constantly growing across different taxa. The use of these regions will represent a powerful molecular tool in future studies.

We used a total of 69 species representing the four tribes of the subfamily Polistinae (Table 1). Specimens were either donated by colleagues, or were in our own collections. These specimens included 33 species from the tribe Polistini, 7 from the Mischocyttarini, 7 from the Ropalidiini and 22 from the Epiponini. We used the Polistini as the outgroup for the Epiponini+Mischocyttarini+Ropalidiini. We used the latter three tribes as the outgroup for the analysis of the Polistini.

We used a total of 932 characters, including 95 morphological and behavioral characters (all morphological characters and character states from Carpenter's studies 25367 examined on pinned specimens for this study; larval and behavioral characters and states from citations given in appendixes 1 and 2); a 488 base pair (bp) fragment of the mtDNA COI gene (appendix 3) 92; a 311 bp fragment of single-copy nuclear flanking sequence of three microsatellite loci (Pbe216AAG [137 bp], Pbe269bAAG [75 bp], and, Pbe411AAT [99 bp] (appendix 4) 5293, and 38 characters representing the different trinucleotides in the repeat region (not their number; appendix 5 78). The repeat regions varied considerably across the four different tribes and across the different subgenera in the tribe Polistini. The coding system for the repeat region was based on presence or absence of each repeat motif and resulted in16 characters for Pbe216AAG, 15 characters for Pbe269bAAG, and 7 characters for Pbe411AAT. For example, the first four characters at Pbe216AAG were GGA, AAA, TAA, GCA, all substituted for AAG within the repeat region 78. We did not count times a specific motif was repeated because this character is so variable its evolutionary history would be unclear.

We extracted DNA using protocol Strassmann I 94. Prior to DNA extractions, we stored all specimens either at -80°C or in 100% ethanol at room temperature. PCR reactions followed protocols as in Wilcox et al. 95 for the mtDNA fragment, and Zhu et al. 78 for the microsatellite loci. Sequencing PCR products were completed directly with thermo sequenase radiolabeled ³³P (Thermo Sequenace radiolabeled terminator cycle sequencing Kit, Amersham Life Science). DNA sequences were run on denaturing polyacrylamide gels, dried and exposed to X-ray film. All new DNA microsatellite sequences were deposited in Genbank under accession numbers AF119623-AF119660, AF120355-AF120455. COI sequences were deposited in Genbank under accession numbers AY382200-AY382265. The aligned DNA matrix is available at TreeBASE http://www.treebase.org/treebase/index.html, submission ID number SN1595.

The sequences for both mtDNA (COI) and for nuclear regions (microsatellite flanking regions) were aligned based on the criterion of maximum parsimony, using the program Malign, specifying a 3:1 change:gap ratio 96. We occasionally adjusted alignments by eye when Malign gave poor results.

We used Paup* 4.0b10 97, Nona 98, and Winclada 99 to construct the phylogeny, with equal weight for all base-pair substitutions (including the mtDNA COI sequence, 100). For the COI gene, the analysis was completed with the use of the three coding positions weighted equally and with the first and second coding positions only. The phylogenetic trees were not in conflict by excluding the third coding position.

We completed the phylogenetic analyses following the principles of maximum parsimony, maximum likelihood and distance analysis. As the overall topology was not in disagreement by applying different algorithms, we are only presenting the results obtained based on maximum parsimony. All parsimony analyses were run using approximate searches, due to the large number of species and characters. The runs consisted of 1000 replications of the parsimony ratchet 101 as implemented in Winclada, with 10% character reweighting and holding one tree; 500 replications at the "default amb-poly+" setting and 500 at "amb = poly-." Resulting trees were read into Nona and swapped on with "max*" (TBR branch swapping). Nona was also used to carry out 1000 random addition sequences as a check. Maximum parsimony trees were first estimated individually for each of the different data sets (morphological and molecular), and then in combination. Gaps were treated as missing characters. Bootstrap analyses with 1000 replications were also run for each data set. Paup* runs gave similar results as those with Winclada and Nona.

Table 2 shows the number of parsimoniously informative characters and constant characters for the each different set of data used in the study, for the 69 species used.