Base-compositional biases and the bat problem. III. The question of microchiropteran monophyly

James M. Hutcheon1,JohnA.W.Kirsch1 and John D. Pettigrew2 1The University of Wisconsin Zoological Museum, 250 North Mills Street, Madison,WI 53706, USA 2Vision,Touch, and Hearing Research Centre, University of Queensland, St Lucia, Queensland 4072, Australia

Using single-copy DNA hybridization, we carried out a whole genome study of 16 bats (from ten families) and ¢ve outgroups (two primates and one each dermopteran, scandentian, and marsupial). Three of the bat species represented as many families of Rhinolophoidea, and these always associated with the two representatives of Pteropodidae. All other microchiropterans, however, formed a monophyletic unit displaying interrelationships largely in accord with current opinion. Thus noctilionoids comprised one clade, while vespertilionids, emballonurids, and molossids comprised three others, successively more closely related in that sequence. The unexpected position of rhinolophoids may be due either to the high AT bias they share with pteropodids, or it may be phylogenetically authentic. Reanalysis of the data with varying combinations of the ¢ve outgroups does not indicate a rooting problem, and the inclusion of many bat lineages divided at varying levels similarly discounts long branch attraction as an explanation for the pteropodid^rhinolophoid association. If rhinolophoids are indeed specially related to pteropodids, many synapomorphies of Microchiroptera are called into question, not least the unitary evolution of echolocation (although this feature may simply have been lost in pteropodids). Further, a rhinolophoid^pteropodid relationshipöif trueöhas serious implications for the classi¢cation of bats. Finally, among the outgroups, an apparent sister-group relation of Dermoptera and Primates suggests that £ying lemurs do not represent the ancestors of some or all bats; yet, insofar as gliding of the type implemented in dermopterans is an appropriate model for the evolution of powered mammalian £ying, the position of Cynocephalus in our tree indirectly strengthens the argument that true £ight could have evolved more than once among bats. Keywords: Archonta; bat monophyly; bat phylogeny; Microchiroptera; molecular evolution

In the two companion papers to this one, Pettigrew & 1. INTRODUCTION Kirsch (1998) and Kirsch & Pettigrew (1998) have The monophyly versus diphyly of bats remains a considered the bias question, respectively presenting DNA contentious issue, despite much marshalling of evidence hybridization elution curves and trees which were based on both sides of the question (e.g. Smith & Madkour not only on whole genome single-copy DNAs but also on 1980; Pettigrew 1986, 1991a,b, 1995; Pettigrew et al. labelled fractions `enriched' for either ATor GC content. 1989; Baker et al.1991b; Simmons et al. 1991; Thewissen While the trees in Kirsch & Pettigrew (1998) may be & Babcock 1991; Simmons 1994). Signi¢cantly, trees regarded as indecisive with respect to bat monophyly, based on DNA data are always at least consistent with they all show, or are consistent with, a remarkable associa- a monophyletic Chiroptera (Adkins & Honeycutt 1991; tion of the representative microchiropteran rhinolophoid Baker et al. 1991a; Mindell et al. 1991; Ammerman & (Rhinolophus philippinensis) and megachiropteran ptero- Hillis 1992; Bailey et al. 1992; Kilpatrick & Nu·ez 1993; podid (Pteropus vampyrus) exclusive of other microbats Stanhope et al. 1993; Kirsch et al. 1995b ; Porter et al. (the noctilionoids, Noctilio albiventris or N. leporinus,and 1996). While the prestige of molecular techniques may Pteronotus parnellii), regardless of which type of label was seem to render such results decisive, the sequencing and used. As rhinolophoids are amongst the most AT rich of DNA hybridization studies conducted to date all su¡er microchiropterans, with up to 70% AT content as from inadequate sampling of the diversity of Chiroptera, compared to the mammalian average of about 60% leaving open the possibility that algorithmic artefacts (Pettigrew 1995), one interpretation of Pettigrew & (such as the `attraction of long branches' (Felsenstein Kirsch's results is that, indeed, such a bias may 1978; Swo¡ord & Olsen 1990)) may be responsible for a¡ect apparent relationships among microbats, if not the joining of microchiropterans and megachiropterans necessarily between these, megabats, and other orders of in most DNA based trees. Alternatively, Pettigrew mammals. (1994, 1995) has pointed out that many, but not all, However, the issue of taxonomic sampling remains microbats share with pteropodids (and some other unaddressed by Pettigrew & Kirsch's experiments: only mammals) a notably elevated AT content, which could four bat species were included in even the largest of their also conceivably bias DNA based topologies in favour of matrices. Therefore, we undertook to extend their whole bat monophyly. genome study (Kirsch & Pettigrew 1998, table 1) to a

Phil. Trans. R. Soc. Lond. B (1998) 353, 607^617 607 & 1998 The Royal Society Received 20 August 1996 Accepted 9 April 1997 608 J. M. Hutcheon and others Hybridization and microchiropteran monophyly total of 21 species, including ¢ve outgroups and represen- very di¡erent ÁNPH index gave results parallel to those tatives of ten bat families. In every case, we were able to using the ÁTmode, so we consider it su¤cient to present subdivide each putative bat lineage at least once, and just results obtained with the latter index here. usually near its midpoint. In this way we hoped to avoid Reciprocal comparisons were corrected for asymmetry the possibility of long branch attraction; and, by inclusion by the method of Sarich & Cronin (1976) to obviate of many outgroups (four of which apparently constitute a systematic experimental error (the `compression e¡ect' of subdivided clade), to reach a reliable conclusion about the Springer & Kirsch (1991), which is mainly due to modal position of the root among bats (if, that is, bats are indeed di¡erences in fragment sizes among tracers), and missing monophyletic). Further, by including several bats whose values were then re£ected from their known counterparts. phyletic positions are fairly well known, any discrepancies Where both members of a pair of reciprocals were unmea- from received opinion would be highlighted and signal sured (ten cases), the entries were estimated by the the need to consider spurious reasons (such as base- additive procedure of Landry et al. (1996). Trees were then composition bias) for the topology obtained. calculated for 21- and 18-taxon subsets of the data, using In the event, our trees did not di¡er from Kirsch & the FITCH program in Felsenstein's (1993) PHYLIP Pettigrew's (1998) as to the position of rhinolophoids, package, employing the global branchswapping, sub- though in all other respects, except possibly the placement replicate, and Cavalli-Sforza & Edwards options and of emballonurids, they were consistent with current views randomly varying the input order of taxa 100 times; about microchiropteran relationships. Additionally, the re£ected or estimated cells were conservatively considered trees suggest that dermopterans are the sister group of as measured once in the tree computations. Fitted path- primates rather than of some or all bats; primates are lengths were correlated with the measured distances in also excluded from the special relationship with megabats. order to obtain an estimate of how well the data However, because our trees only included putative archon- conformed to the assumption of additivity. The 21- and tans as eutherian outgroups, we do not claim to have 18-taxon trees were validated by Krajewski & Dickerman's resolved the question of bat monophyly. Nevertheless, the (1990) adaptation of bootstrapping for distance data (a results with respect to rhinolophoid microbats mean that technique for exploring measurement error), generating a either the e¡ect of high AT content on molecular trees is consensus of 1000 pseudoreplicate trees in each case; and real, or a radical rethinking of bat relationships is in the 21- and 18-taxon topologies were further tested for orderöin particular, with respect to the monophyly of stability of the underlying matrices by the jackknife on Microchiroptera and the characters which de¢ne that taxa for weighted trees of Lapointe et al. (1994). For the taxon. If microbats are indeed paraphyletic, as our trees jackknives, both `all single' and `500 random' deletions suggest, then the present subordinal dichotomy within (drawn proportionately from among the possible combi- Chiroptera should be abandoned. nations of single and multiple deletions) were performed, calculating the average pathlengths recovered over the pseudoreplicates in each case. For the 21-taxon tree, we 2. MATERIALS AND METHODS also jackknifed separately on all possible combinations of Methods for puri¢cation of DNA, preparation of the ¢ve outgroups (including none), with the suite of bats extracts for radiolabelling with 125I and hybridization, held constant; an average consensus was then calculated and evaluation of hybrids were as outlined in earlier from the pathlengths on FITCH trees corresponding to papers (Kirsch et al. 1990; Bleiweiss et al. 1994), except all such deletions, as for the jackknives on the entire that the single-copy fractions were separated at a higher matrix or the 18-taxon subset, omitting the three unla-

Equivalent-C0t (1980^2260 rather than 1130), and belled taxa. For both the bootstraps and jackknives, each amounts of driver DNA were reduced to 25 or 13 mg from pseudoreplicate matrix was separately symmetrized and 50 mg. Eighteen of the 21 species examined were labelled completed prior to tree construction. and over 1000 hybrids were prepared, with tracer:driver Finally, the data were tested for phylogenetic (or other) ratios of ca. 1:500. structure by a randomization test (Kirsch et al. 1995a). This A matrix was assembled from two or more `runs'of up to test produces a `z score,' which essentially states how many 25 hybrids with each of the 18 tracers, the di¡erences (Á's) standard deviations from the mean sum-of-squares for in melting temperatures being calculated from 56 8C with FITCH trees based on randomized data lies the sum-of- reference to 2^8 homoduplexes per label (averaged across squares for the tree calculated from the unrandomized runs) and indexed as ÁTmodes. Modes are not ordinarily matrix. Because outgroups may render this test too considered appropriate for very distant comparisons liberal, we performed this test at two levels: on the full among mammals due to a marked low temperature peak matrix (but holding the outgroup opossum values with which the true mode may be con£ated, and which constant) and on the matrix without any outgroups. may be caused by the presence of many poorly matched paralogous sequences (Fox & Schmid 1980): usually the height, but not location, of this secondary peak is corre- 3. RESULTS lated with distance (Kirsch et al. 1995a). However, bat Figure 1 shows representative thermal elution curves hybrids do not seem to show such a peak, or only rarely, obtained with the labelled rhinolophoid Hipposideros galeritus, and empirically a single peak of variable position was and illustrates the level of discrimination generally found in found in its place when plotting curves involving distantly our experiments. Rhinolophus philippinensis (a member of the related taxa; apparently this is the true mode, as it seemed same microchiropteran superfamily as the homologous to be in Pettigrew & Kirsch's (1998) experiments. More- species) is approximately intermediate between the homo- over, Kirsch & Pettigrew's (1998) trees employing the logues and the depicted molossid microbat Molossus sinaloae;

Phil. Trans. R. Soc. Lond. B (1998) Hybridization and microchiropteran monophyly J. M. Hutcheon and others 609

Figure 1. Representative thermal elution curves for hybrids with the labelled rhinolophoid microbat Hipposideros galeritus (replicate homologues at right). Each elution has been divided by total counts and expressed as a percentage of the total eluted for that hybrid to show the extent of hybridization as well as distribution of counts. The Hipposideros tracer was one of the few bat labels to show any evidence of a low temperature peak. most other non-rhinolophoid microbats were about as Figure 2 con¢rms the association (here, with 100% distant from rhinolophoids as was Molossus, but the bootstrap support) of Rhinolophus and Pteropus found by outgroups (such as the dermopteran Cynocephalus variegatus, Kirsch & Pettigrew (1998), with representation of their shown here) were still more distant. However, pteropodids lineages here increased from one rhinolophoid species to (e.g. Pteropus vampyrus in ¢gure 1) were markedly closer to three (representing as many families) and from one to the homologues than were non-rhinolophoid microchirop- two pteropodids. All other microchiropterans group terans, and reciprocal experiments were always consistent together with high bootstrap support (88%), with the with the ordering depicted in ¢gure 1. three noctilionoids separated from the rest (100%

Table 1 presents the raw ÁTmodes for the 18 labels, with support). Emballonurids are the sister group of molossids, corrections for asymmetry listed at the bottom; and also with moderate (83%) support; and vespertilionids are (on the third lines of cells) the values used to construct the more weakly positioned (69% support) as part of a larger trees of ¢gures 2 (of all 21 taxa) and 3 (of the 18 labelled group including emballonurids and molossids. In ¢gure 3, species only) after symmetrization, re£ection of 94 cells, based on just the 18 labelled taxa (47 cells or 15% of the and estimation of ten missing pairs (i.e. 26% of the possible total comparisons were re£ected after symmetrization), comparisons were re£ected or estimated for calculation of the molossid^emballonurid relationship has 91% ¢gure 2). The correlation (r) of ¢gure 2 ¢tted pathlengths support). However, the relationship of vespertilionoids with these distances is 0.96, indicating near perfect addi- with molossids-plus-emballonurids in ¢gure 3 is poorly tivity of the table 1 values; rˆ0.98 for the 18-taxon subset supported by the bootstrap (at 48%) and not by the of ¢gure 3. Both correlations are highly signi¢cant average consensus on the 500 random deletion jackknives (p50.01) even with a very conservative estimate of degrees (where noctilionoids occupy that position; the intervening of freedom (10 and 9, respectively). In both ¢gures 2 and 3, internode is therefore shown as a thin line). Within any bootstrap numbers have been shown at the nodes (except familial or superfamilial group of bats, recovered relation- for the root node, which was ¢xed by de¢nition). The two ships are as currently accepted (e.g. Koopman 1994), and thin lines in ¢gure 3 indicate discrepancies between the bootstrap support for each node is 98% or better in both FITCH and jackknife trees (see below). ¢gures 2 and 3. Moreover, comparisons using additional

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Phil. Trans. R. Soc. Lond. B (1998) Hybridization and microchiropteran monophyly J. M. Hutcheon and others 611 5 4 0 0 2 2 7 0 7 3 1 8 2 7 6 4 5 8 5 7 8 6 ...... 0 na na na na na na na na na na na na na 1 2 4 0 3 1 0 0 0 8 0 2 2 1 2 2 2 2 2 2 1 2 9 2 1 3 5 3 6 3 8 0 1 1 4 4 0 5 7 0 6 2 0 6 ...... 0 na na na na na na na na na na na na na 3 3 2 5 8 2 2 2 2 0 0 2 2 2 1 1 2 2 2 2 2 2 2 8 1 3 9 7 1 1 8 0 3 1 1 6 5 9 0 6 4 6 0 8 ...... 0 na na na na na na na na na na na na na 3 7 2 3 2 3 2 2 3 0 8 2 2 2 2 2 2 2 2 2 1 5 3 3 3 3 2 3 3 3 3 2 9 9 9 5 5 6 8 5 9 2 8 0 0 4 3 9 5 4 2 2 9 0 0 1 1 1 3 6 1 7 1 5 5 0 0 0 9 1 1 3 / 3 1 8 5 5 1 1 9 0 / 2/ 2/ 6/ 6/ 8/ 1/ 6/ 7/ 7/ 0/ 0 4 4 6 3 5 1 5 1 4 5 2. 4. 1. 1. 0. 2. 2. 1. 3. 4. 4. 2. 2. 3. 3. 2. 3. 1. 1. 2. 3. 2. 0. .9 na na 2 8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 2 4 4 3 3 3 2 3 3 3 2 2 4 3 1 6 1 2 7 0 4 3 6 2 9 3 3 2 4 9 4 3 6 1 8 /1 3/ 9/ 7/ 2/ 0/ 4/ 0/ 4/ 1/ 1/ 5/ .5 .9 .2 .4 .4 .3 .0 .9 .9 .0 .3 .1 .1 .1 .5 .4 .4 .2 .9 .0 .1 .3 .2 0 98 1 7 3 2 3 3 3 3 5 3 9 6 7 na 18 17 19 83 21 23 20 21 24 24 22 21 23 23 21 23 22 20 23 21 20 0. 3. 0. 0. 0. 0. 1. 0. 0. 0. 0. 0. 2 2 4 2 2 4 4 4 2 2 5 1 5 3 2 4 5 1 3 6 6 5 7 0 1 0 1 2 8 5 2 1 1 3 1/ 0/ 6/ 7/ 7/ 9/ 5/ 7/ 1/ 0/ .9 . . .2 .6 .9 .8 .5 .0 .3 .1 .8 .7 .1 .4 .7 .6 .7 .3 .7 .9 0 02 na 2 na 4 4 2 2 5 7 6 9 3 2 1 22 85 21 2 21 22 22 22 22 2 21 20 21 22 22 14 22 22 22 22 14 1. 0. 0. 0. 0. 0. 1. 0. 0. 0. 0. 2 2 3 2 2 4 4 3 2 2 6 2 4 4 5 7 9 8 9 7 9 4 0 3 3 0 3 5 6 2 6 5 0 18 9/ 6/ 6/ 3/ 4/ 7/ 4/ 7/ 0/ 0/ .3 . . .2 .6 .5 .5 .1 .5 .1 .8 .2 .2 .7 .0 .6 .5 .2 .6 .8 .0 0 9 na na 2 3 .5 .6 .2 .5 .6 .4 .9 .2 .2 .4 26 24 21 85 2 22 20 21 24 2 24 22 21 23 15 20 23 24 21 16 23 0. 2 0 0 0 0 0 1 0 0 0 2 2 3 4 3 6 3 6 4 9 4 6 1 9 1 8 5 9 1 8 9 8 1 5 2 9 3 1 2 2 0 0 46 /1 6/ 0/ 4/ 5/ 7/ 7/ 5/ 9/ 1/ .5 . . .8 .2 .1 .2 .8 .7 .4 .8 .2 .3 .9 .5 .4 .9 .4 .8 .8 .0 0 9 na na 4 4 .3 .2 .6 .5 .7 .2 .2 .6 .2 na 24 23 22 17 18 2 84 23 25 24 23 2 22 23 18 23 22 25 23 19 25 0. 0 0 1 0 0 1 1 1 0 2 3 5 6 4 3 /2 /2 /4 /4 /7 /6 /4 /7 /2 /4 0 30 3 65 44 47 69 98 75 17 73 52 07 20 98 5 4 95 73 2 02 3 . . 0 30 na na 47 24 34 32 51 60 39 88 34 3. 4 1. 1. 2. 2. 2. 2. 4 5. 2. 1. 3. 2. 1. 9. 9. 2. 1. 9. .0 9. 2 2 2 2 2 2 2 2 8 2 2 2 2 2 2 2 2 1 0. 1. 0. 0. 0. 0. 0. 0. 0. 0. 0 9 5 7 /2 /2 /4 /2 /2 /4 /4 /4 /2 /2 1 68 1 76 69 11 18 30 23 02 05 08 06 87 61 84 77 13 94 6 24 6 . . 0 31 na na 29 96 40 12 20 33 64 91 81 2. 3 1. 1. 1. 1. 2. 2. 3 0. 0. 6. 2. 0. 2. 1. 1. 2. 0. 6. .0 6. 2 2 2 2 2 2 2 2 2 1 1 8 2 2 2 2 2 2 2 1 1. 1. 0. 0. 0. 0. 0. 0. 0. 0. 2 2 3 2 2 4 4 4 2 2 4 4 3 3 4 7 7 5 1 8 3 0 1 2 0 7 6 2 3 2 5 1 3 7 5 6 8 91 24 47 24 8 6 5 4 9 1 4 2 0 7/ 2/ 2/ 3/ 6/ 6/ 8/ 2/ 9/ 3/ . . 0 na 0 na 0 8 0 1 2 1 9 0 5 3. 2. 1. 3 1. 2. 3. 2. 3 8. 5. 9. 2. 0. 2. 2. 2. 6. 6. 3. 1. .0 2 2 2 2 2 2 2 2 2 8 2 2 2 2 2 2 2 1 1. 0. 0. 0. 0. 0. 0. 0. 1. 0. 2 2 4 4 5 4 8 2 4 2 4 1 8 8 3 6 4 0 8 8 6 9 5 8 7 8 6 5 6 2 5 0 9 5/ 1/ 2/ 4/ 3/ 8/ 3/ 9/ 4/ .8 . . .9 .9 .4 .5 .5 .3 . .2 .2 .0 .1 .2 .7 .6 .0 .3 .7 0 95 na 0 na na 4 8 4 5 4 5 7 3 2 3 1 24 22 21 22 23 24 23 2 22 21 2 22 23 22 20 23 86 23 21 0. 2 1. 0. 0. 1. 1. 2. 0. 1. 1. 2 2 4 2 7 5 4 8 2 4 3 5 / / / / / / / / / / 24 4 1 44 85 44 00 51 86 70 27 37 79 30 99 13 08 60 39 52 73 61 32 ...... 9 35 na na 66 12 47 88 76 01 56 22 22 3 4 25 24 22 22 24 25 23 2 22 24 24 22 24 2 25 23 22 23 13 12 24 23 0. 0. 0. 0. 0. 0. 0. 1. 0. 0. 0. 4 2 3 3 2 4 3 4 3 2 6 6 4 9 7 0 5 7 3 7 9 6 2 1 5 8 5 6 2 7 0 8 2 6 /1 47 /1 2/ 3/ 5/ 1/ 1/ 8/ 7/ 2/ 6/ 8/ .4 .6 .1 .5 .0 .6 .1 .3 .8 .0 .5 .7 .2 .3 .8 .2 .7 .8 .8 .9 .0 .5 .3 .2 9 .3 .7 .2 .0 .1 .4 .6 .5 .4 .1 na na 24 23 24 23 24 22 22 22 22 24 23 22 23 23 22 23 23 22 21 23 21 20 23 22 0. 0 0 0 0 0 0 0 0 0 0 6 7 7 7 3 5 0 7 2 9 0 0 3 7 2 1 8 8 2 7 9 5 5 /2 /2 /4 /4 /8 /6 /4 /8 /2 /3 3 8 6 8 5 1 1 5 4 7 0 4 .6 . . .9 .4 .5 .1 .5 .9 .3 .0 .3 .7 .4 .0 .9 .7 .0 .5 .0 .4 .3 92 na na 2 2 .4 .4 .9 .8 .0 .9 .4 .2 .6 .2 23 22 21 21 23 24 22 2 22 24 23 21 21 23 2 21 21 22 21 20 23 23 0. 1 1 0 0 2 1 1 1 1 1 2 7 /4 /2 /2 1 /5 /2 /2 /2 /2 1 83 2 4 56 49 16 19 33 16 44 51 72 / 63 38 44 43 63 72 80 94 50 59 75 / . . na na 24 23 27 60 10 66 11 01 5. 5. 2. 2. 2 5. 6. 3. 2. 5. 3 5. 2. 2. 5. 2. 1. 2. 3. 0. 5. 4. .8 na na 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0. 0. 0. 0. 0. 0. 0. 0. 2 2 2 2 2 2 2 3 2 2 2 5 4 0 0 7 2 9 3 0 6 4 8 4 8 7 7 8 0 5 0 0 4 2 9 1 2 1 2 5 6 9 8 3 8 2 5 5 6 9 0 1 0 2 7 8 8 0 0 1/ 8/ 0/ 2/ 4/ 3/ 6/ 4/ 9/ 3/ 9/ . na 1 2 3 2 7 1 2 8 0 0 7 0. 8. 4. 7. 7. 4. 4. 7. 7. 4. 4. 7. 5. 1. 4. 7. 5. 5. 7. 4. 7. 8. .4 1 2 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 2 2 1 2 1 1 1 2 0. 1. 0. 1. 0. 1. 2. 0. 1. 1. 0. 2 4 2 4 9 0 6 4 7 8 9 6 7 8 8 9 8 6 6 3 1 7 6 6 7 5 1 8 4 1/ 5/ 3/ 8/ . . . . .5 .8 .9 .6 .8 .1 . . . . .4 .8 91 na na na na na na 5 na na 3 3 4 3 3 3 2 3 0 1 1 25 23 2 23 25 25 24 2 24 22 2 2 0. 2 2 2 2 0. 0. 0. 0. 2 2 4 4 4 6 4 7 2 4 7 0 / / / / / / / / / / 93 8 8 31 68 00 61 18 58 26 85 02 40 94 55 31 30 87 46 88 92 47 79 ...... 9 09 na na 41 09 97 76 98 33 04 16 37 4 4 24 25 24 24 24 24 23 23 2 23 25 24 23 24 24 24 23 2 23 22 24 24 0. 0. 0. 0. 0. 0. 0. 0. 1. 0. 0. 2 2 2 2 2 3 2 2 3 3 4 7 1 3 8 6 3 5 9 1 7 0 6 9 2 0 3 1 5 4 1 9 /1 09 /1 /1 5/ 6/ 4/ 3/ 2/ 0/ 4/ 2/ .0 .3 .4 . .3 .4 .5 .7 .2 .1 .6 .0 .7 .6 .4 .4 .5 .8 .4 .3 .0 .9 .3 2 na 4 .8 .3 .7 .3 .4 .5 .0 .5 na na na 18 21 25 21 25 23 19 20 24 22 19 19 23 24 20 21 23 25 19 23 2 19 21 1. 0 2 0 0 0 1 0 0 ed 2 2 4 1 2 0 4 0 5 7 7 1 2 6 7 4 0 u 3 5 8 9 6 7 8 0 8 33 /1 n 2/ 1/ . . . . .2 .4 . .8 .1 . . . . .8 .0 1 na na na na na na na na na 7 5 7 3 4 5 2 3 2 ti .2 .7 na 21 24 2 21 25 20 24 2 2 1. 2 2 2 2 2 2 0 0 n Co is is s er s e is nu ens s ct r a i tu a s n us n 1. e i in yx ia o us el nt to ny gu at us a ll ou io it ph il t oa er us e us pp t ph ve en io fu li at n ne ho o ne lo um oz ur li ss al pt ic is no ph s r h tt re il li bi ec ci ic bl nc lo ot es no in co oz mo ph to ro ct y t i ae bi ma s na pa al ku lu ho phi c pl a rr e ac co S Ta Rh Mo Pt No S My Ep T Rh Mi Ch co

Phil. Trans. R. Soc. Lond. B (1998) 612 J. M. Hutcheon and others Hybridization and microchiropteran monophyly

Figure 2. FITCH tree of relationships among 16 species of bats and ¢ve outgroups, from data of table 1 (third lines of cells). Numbers at nodes (except for the root) are bootstrap replicates supporting those clades. Branch lengths are drawn approximately to scale, and are based on the averages observed over 1000 bootstrap pseudoreplicate trees. Animal ¢gures redrawn after several sources, but primarily Hill & Smith (1980). species as drivers only (data not shown) always gave Because determination of the intrachiropteran root is distances from a confamilial tracer which were less than critical to the question of the a¤nity of rhinolophoids, those to other families; the use of single species to repre- we also carried out a jackknife on the full (21 taxon) sent families or subfamilies therefore appears justi¢ed. data set involving deletions just of outgroups. Trees Didelphis generally rooted the trees between bats and the calculated from a series of subsets of the data where, four non-bat eutherians. As expected, the two primates collectively, all possible combinations of the ¢ve non- were the closest pair among these latter four; less expected bats were excluded (30 trees), did not materially alter was the sister-group relation of Cynocephalus to the intrabat relationships. Elimination of all ¢ve outgroups primates, but Tupaia was only very weakly associated with similarly gave the same topology among bats alone as the other three, judging by the bootstrap percentages (in is shown in ¢gure 2, although obviously the root of fact, support for placing Tupaia with the bats is 51% for this outgroupless tree could not be determined. the 18-taxon matrix). The last node is also the single However, we note again that bootstrap support for the outgroup relationship which was unstable in the jack- placement of Tupaia on ¢gures 2 and 3 is low, and that knives, though only on the 18-taxon subset of table 1. Both the average consensus tree for random deletions on the jackknife average consensus trees on the 21-taxon set 18-taxon subset placed the scandentian as sister to the placed Tupaia with non-bats, as did the average consensus chiropterans. of single deletions on the 18-taxon subset; random Results of the randomization tests were highly signi¢- deletions among the 18 taxa put the scandentian with cant: the z score for the full table 1 matrix (with the bats, however. Thus, with respect to the eutherian taxa outgroup opossum distances held constant) was 11.30; for studied here, neither Primates nor the £ying lemur the 16-taxon matrix lacking all outgroups it was 9.76. A z appear to be more nearly related to some or all bats than score of about 2 would be considered signi¢cant at the latter are among themselves (a result consistent with p50.05, assuming a normal distribution of the rando- ¢g. 7a in Kirsch et al. (1995b), where bats grouped together mized sums-of-squares, which they have. Therefore, these and with ungulates rather than with Perodicticus, Rattus, or results strongly suggest that the ÁTmode index provides Scalopus); and whileTupaia is clearly not a primate, its posi- discrimination among the studied taxa (i.e. that the data tion should be considered unresolved by our experiments. have phylogenetic or other structure).

Phil. Trans. R. Soc. Lond. B (1998) Hybridization and microchiropteran monophyly J. M. Hutcheon and others 613

Figure 3. FITCH tree of relationships among 13 species of bats and ¢ve outgroups, from table 1 data on labelled taxa only. Numbers at nodes (except for the root) are bootstrap replicates supporting those clades. Thin lines indicate relationships not supported in average consensus trees of both single and random deletion jackknives: single deletions put Tupaia with non-bat eutherians (as shown), while random deletions placed the scandentian with bats and interchanged the positions of vespertilionids and noctilionoids. Branch lengths are drawn approximately to scale, and are based on the average consensus single-deletion jack- knife tree. Rhinolophoidea (Koopman 1994)), on the other hand, 4. DISCUSSION might provide additional basal chiropteran subdivisions; (a) Analytical considerations but the rhinolophoid branch is already fairly evenly subdi- Our results raise several issues of an analytic as well as a vided in ¢gures 2 and 3, as are all familial or superfamilial phylogenetic nature. First, it no longer appears possible to lineages within the non-rhinolophoid microbats. More- attribute the linking of the rhinolophoid and pteropodid over, in a recent DNA hybridization study of 26 found by Kirsch & Pettigrew (1998) solely to long branch hummingbirds, Bleiweiss et al. (1997) showed that the attractionöor at least, if it is due to such an algorithmic basic structure of their tree was unaltered even when the artefact, there is little more that can be done to alleviate number of ingroup taxa was reduced to six. For FITCH that problem. Dobsonia and Pteropus are about as numeri- analysis of DNA hybridization data, at least, the long cally distant from each other as are any two pteropodids branch attraction problem may have been overstated. examined (Kirsch et al. 1995b), even though the relatively The position of the root within Chiroptera recovered slow evolving Nyctimene and Paranyctimene together seem to here is thus more than an artefact of poor taxonomic comprise the sister group to other pteropodids (Kirsch et sampling among bats; but as Marshall (1991) and Kirsch al. 1995b, ¢gs 2, 3, and 6); inclusion of additional pteropo- et al. (1995c) have shown, when ingroup taxa are separated dids would not provide much by way of divisions further by short internodes, experimental error among ingroup^ down the branch connecting Dobsonia and Pteropus to the outgroup distances may signi¢cantly distort ingroup node joining them with rhinolophoids. In fact, when we relationships. Here, a shift of the intrabat root by only `sutured' the table 1 data with a matrix among 19 ptero- one node is all that would be required to restore micro- podids (Kirsch et al. 1995b, table 2 in that paper), the chiropteran monophyly. Yet we took care to include a suite resulting FITCH tree did not di¡er for shared taxa from of outgroup species, most of which were themselves likely ¢gure 2, except thatTupaia was sister to the bats. Inclusion to be interrelated apart from the ingroup, as seems good of craseonycterids, nycterids, or rhinopomatids (which are practice in molecular systematics (Smith 1994). The single other taxa sometimes placed with Emballonuroidea or and random deletion jackknife analyses on table 1 (21- and

Phil. Trans. R. Soc. Lond. B (1998) 614 J. M. Hutcheon and others Hybridization and microchiropteran monophyly

18-taxon subsets) show that incorrect rooting due to short ¢gures 2 and 3 do seem to exclude the possibility that and uncertain basal internodes is probably not the expla- £ying lemurs are by themselves sister to either group of nation for our surprising phylogeny, and the more explicit bats, we caution that the internodes among the four outgroup-deletion experimentsösequentially removing eutherian outgroups are very short, the lineages are not all possible combinations of the ¢ve outgroups from table generally subdivided, and some of our experiments with 1öalso failed to change the position of the intrabat root. fractionated DNAs (Kirsch & Pettigrew 1998; Pettigrew Moreover, with all outgroups eliminated, ingroup & Kirsch 1998) support a di¡erent interpretation where topology remained unaltered, although of course the root megabats and the colugo are much closer; many morpho- among bats was then undetermined. Nevertheless, inclu- logical and neural data also support the latter association sion of representatives from additional eutherian orders (Pettigrew 1995). There has, however, been some dispute could conceivably produce di¡erent relationships among recently regarding the association of Dermoptera with the taxa of ¢gure 2, but then problems of resolution other presumptive archontans. Beard (1993) noted that would doubtless be exacerbated due to the subtleties of super¢cial morphological similarities based primarily on partitioning already very short basal internodes. £ying/gliding adaptations lead to the `unnatural assem- However, our results are not likely to be incorrect because blage' of Volitantia, a cohort comprised of all bats and of poor discrimination by the thermal stability index colugos. Beard argued that the patagia of these two taxa chosen: randomization tests on the matrix were highly are, in fact, convergent and that dermopterans are signi¢cant for structure. It may be objected that the subdi- connate with primates. Several palaeontological and mole- vision of bat clades subverts this conclusion with respect to cular studies (see Beard 1993), as well as a number of the intrachiropteran root because the tests are therefore neural apomorphies that are uniquely shared between too liberal, but we performed another randomization test primates and colugos (Pettigrew et al. 1989) or speci¢cally on single representatives of each major chiropteran lineage between tarsiers and colugos (Rosa et al. 1996), have also (six taxa), leaving only very short basal internodes and no suggested this relationship, which if true has interesting subdivisions along the bat lineages, and still obtained a implications for the evolution of £ight; and, indirectly, for highly signi¢cant result (z scoreˆ5.03). the question of bat monophyly. It is, however, possible that no combination of Most current scenarios for the evolution of chiropteran outgroups would produce a di¡erent placement of the £ight suggest a move from an arboreal, insectivorous rhinolophoids. If it is correct that a shared AT bias progenitor, through a gliding phase, to true powered accounts for the observed a¤nity of rhinolophoids with £ight (cf. Smith 1977; Hill & Smith 1980; Norberg & pteropodids (or, alternatively, the `repulsion' of pteropo- Rayner 1987). None of the presumed transitional stages dids from proximity to Primates), and that a bias is likely have yet been demonstrated in the fossil record, and the to be manifested as an apparently slower rate of evolution earliest known chiropteran fossils with reasonably (Pettigrew & Kirsch 1998), then AT-biased taxa will complete postcranials appear to have been wholly inevitably terminate relatively short branches and may be capable of powered £ight (e.g. Jepsen 1966, 1970). segregated apart from other taxa even by a FITCH However, assuming that the gliding-to-£ight narrative is computation, despite the fact that FITCH is relatively correct, and that the particular implementation of gliding forgiving of apparent rate nonuniformities. Interestingly, in Cynocephalus (i.e. all limbs, the ¢ngers, and the tail in Pierson's ¢g. 3 (1986, opposite p. 21), a midpoint rooted being involved in providing a lifting surface) provides a FITCH tree based on microcomplement-¢xation data, good model for the early stages of evolution of powered Pteropus formed a cluster with both emballonuroids and £ight, then the molecular evidence for removal of £ying rhinolophoids. Another, more recent, serological study lemurs from proximity to bats suggests that true £ight (Schreiber et al. 1994) seems to align both megadermatids could have evolved more than once among mammals. and phyllostomids with megabats, but this investigation That is to say, because no living glider seems to represent was based entirely on one-way comparisons using anti- the ancestor of some or all bats, gliding itself (and of the human sera. Thus, the attempt to ¢x a root among bats dermopteran variety) must have evolved at least twice; may be intrinsically doomed (here, because of the attrac- and if twice, then why not three times? For proponents tion of short (not long!) branches), and in general we do of bat diphyly, this argument should be encouraging, not see that the situation is much better for parsimony as because it removes some of the stigma of positing opposed to distance analyses (cf. Janke et al. (1994, 1996) dual evolution of the complex £ight mechanisms of for examples of unlikely associations among the mamma- Chiroptera. lian infraclasses and cohorts). What also seems clear from our results is that the rhino- lophoid^pteropodid association is `real,' whether because (b) Phylogeny and character evolution these taxa share a high AT: GC ratio or because they are Whatever the veracity of the within-bat root, our trees in fact specially related. Concerning the ¢rst possible do appear to support bat monophyly, but it should be cause, once again we must stress that only additional tests emphasized that only a few eutherian outgroups were with GC rich labels extending Pettigrew & Kirsch's (1998) included; the results really say nothing even about the experiments to fractions which would completely obviate monophyly of Cohort Archonta, much less indicate the the AT bias, but including a wider range of species (as results which would be forthcoming with respect to bats here), can determine if the rhinolophoid^pteropodid had representatives of additional orders been included relationship is entirely due to a biochemical artefact. In and controls for base composition bias been used (e.g. had this regard, we note that Porter et al. (1996) found a the experiments been repeated with GC-enriched labels; particular association of the rhinolophoid Megaderma with but cf. ¢gs 1E^H in Kirsch & Pettigrew (1998)). While pteropodids, a result reminiscent of ours.

Phil. Trans. R. Soc. Lond. B (1998) Hybridization and microchiropteran monophyly J. M. Hutcheon and others 615

On the other hand, anatomical studies suggest that rhinolophoid^pteropodid alliance would require the emballonuroids are phyletically near Rhinolophoidea, while reversal (or independent acquisition) of more than 50 our DNA hybridization trees place Emballonuridae nearest characters at the node joining these taxa, in systems to molossids; Koopman (1994), for example, considers ranging widely from brain to skin to reproduction (see Emballonuroidea and Rhinolophoidea to constitute his Pettigrew 1995, table 1). In the case of the brain characters, microchiropteran infraorder Yinochiroptera, although one can make functional arguments for and against losses Simmons (1998) questions the monophyly of emballonur- or shared gains, like those made in the case of sonar; while oids, and this is also an implication of Pierson's (1986) in other cases, such as the striated pilo-erector muscle data. Otherwise, all interbat relationships shown in ¢bres of rhinolophoids compared with the smooth pilo- ¢gures 2 and 3 herein agree closely with conventional erector muscle ¢bres of the pteropodids, the needed views, a circumstance which makes the anomalous place- reversal is puzzling whether one can provide an explana- ment of rhinolophoids (and, to a lesser extent, of tion or not. A general treatment of the scenarios that could emballonurids) the more striking. have given rise to a monophyletic clade including both It is possible to make a case in support of our association pteropodids and (all or some) microbats must include the between pteropodids and rhinolophoids on zoogeographic following two broad possibilities: (1) where the microbat grounds, as outlined below, but there are numerous anato- general condition is ancestral, or (2) where the pteropodid mical characters that must have undergone homoplastic condition is thought to be ancestral (`deaf fruit bat' and changes to explain this association. Most obviously, `blind cave bat' scenarios respectively, as discussed in Petti- uniting rhinolophoids and pteropodids implies either the grew et al. (1989)). separate evolution of echolocation in rhinolophoids and Regarding more classical craniodental and skeletal other microbats or the loss of this adaptation in Ptero- characters, we note here only that at least one student of podidae. Laryngeal sonar is a sophisticated form of bat anatomy (Sige¨ 1993) has suggested a phylogeny echolocation found only in Microchiroptera (in all 17 broadly similar to that implied in our ¢gures 2 and 3, extant families as well as in some extinct taxa, the latter deriving pteropodids along with rhinolophoids and some inference being based upon radiographic evidence of the other microchiropterans from archaeonycterids, sepa- associated cochlear specialization (Novacek 1985)). This rately from a clade that includes vespertilionoids and adaptive complex is lacking in pteropodids, although one which apparently originated from paleochiropterygids. genus (Rousettus) has developed a primitive form of echolo- However, some aspects of Sige¨ 's phylogram (e.g. inclusion cation involving tongue clicking, so it is necessary to of noctilionoids in a clade with megabats) are falsi¢ed by explain the absence of such a valuable ability in the ptero- our data (see also ¢g. 6 in Kirsch et al. (1995b)). podids (apparent close relatives of the echolocating Finally, a weak case that our tree might be phyletically rhinolophoids in our DNA hybridization trees), which true could be made from distributional data. It appears have comparable navigational requirements in a nocturnal probable that megachiropterans originated in Asia or or crepuscular volant niche. The lack of sonar in ptero- the Australo-Paci¢c region (Ducrocq et al. 1993), and podids is particularly di¤cult to explain if one considers Bogdanowicz & Owen (1992) concluded that rhinolophids the arguments of Speakman et al. (1989). These authors sensu stricto likewise had a southeast Asian origin. In a later suggest that volant organisms have a special advantage in study, Bogdanowicz & Owen (1998) were unable to decide the energetically costly production of sonar pulses, about the centre of origin of hipposiderids; but Hand & because they can achieve e¤ciencies not available to Kirsch (1998) concluded from a mapping of geographic terrestrial organisms by co-opting the same muscles for distributions on their cladogram of Hipposideridae that use both in £ight and in sonar production. If one posits Australasia was a likely ancestral area for that family, a that pteropodids are ancestral to rhinolophoids, then result we have since con¢rmed with Bremer (1992) ances- rhinolophoid sonar must have evolved independently of tral area reconstruction (J. M. Hutcheon, unpublished laryngeal sonar in other microbats, a remarkable paral- data). Of course, all such historical^zoogeographic lelism given the extraordinary sophistication of the conclusions depend greatly on the suite of taxa examined, processing machinery required and the unique elaboration and even more on the reliability of trees relating those of the cochlea found in all microbats. On the other hand, if taxa; but more broadly based considerations of bat distri- pteropodids are derived from rhinolophoids, then one has butions (Hershkovitz 1972; Pierson 1986) have also led to to explain the loss of sonar and its subsequent reacquisition the conclusion that some at least of the extant microbat (in Rousettus) in a primitive and vastly simpli¢ed form. families originated in Gondwanaland. As the case for a Since much is known about microbat sonar, it is possible southeast Asian or Gondwanan origin seems strongest to argue for and against these di¡erent scenarios for the for rhinolophoids and pteropodids, and some of evolution of sonar in pteropodids and rhinolophoids in a the included taxa have similar current distributions as cogent way that emphasizes the di¤culties facing our well as congruent subsidiary geographic moieties (cf. attempts to reconcile the phylogenetic con£ict demon- Bogdanowicz & Owen 1992; Kirsch et al. 1995b), it is strated here. But other systems also show marked perhaps not entirely fanciful to adduce historical zoogeo- di¡erences in pteropodids when compared with graphy in support of a rhinolophoid^pteropodid rhinolophoids. It is worth pointing out that examination relationship. However, the same sort of argument of these systems gives rise to a long list of fundamental might be made for a proposed megabat^colugo-primate di¡erences between pteropodids and microbats, each of association. which would have to be reversed were our pteropodid^ As for the classi¢catory and nomenclatural implications rhinolophoid association real. Space does not permit of a pteropodid^rhinolophoid association, these would discussion of all of these di¡erences, but the most obviously include submergence of Megachiroptera

Phil. Trans. R. Soc. Lond. B (1998) 616 J. M. Hutcheon and others Hybridization and microchiropteran monophyly within a subordinal (or ordinal, if bat monophyly be Felsenstein, J. 1993 PHYLIP, phylogenetic inference package, program disproved) group that includes at least Pteropodidae and and documentation, version 3.5c. Seattle, WA: University of Rhinolophoidea. Koopman's (1994) Yinochiroptera is an Washington. available name, although that taxon might need to be Fox, G. M. & Schmid, C. W. 1980 Related single copy sequences further rede¢ned to exclude Emballonuridae (Pierson in the human genome. Biochim. Biophys. Acta 609, 349^363. Hand, S. J. & Kirsch, J. A. W. 1998 A southern origin for 1986; Simmons 1998; this paper). However, as we have the Hipposideridae (Microchiroptera)? Evidence from the not yet examined other putative yinochiropteran families Australian fossil record. 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