Multiple Sources of Character Information and the Phylogeny of Hawaiian Drosophilids Richard H. Baker; Rob DeSalle Systematic Biology, Vol. 46, No. 4 (Dec., 1997), 654-673. Stable URL hitp://links jstor-org/siisici=1063-5157% 281997 12% 2946%3A4% 3C654%3AMSOCTA%3E2.0,CO%3B2-I Systematic Biology is currently published by Society of Systematic Biologists ‘Your use of the ISTOR archive indicates your acceptance of JSTOR’s Terms and Conditions of Use, available at hup:/www,jstororglabout/terms.hml. ISTOR’s Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at hutp:/wwwjstor.org/journals/ssbiol html Each copy of any part of @ JSTOR transmission must contain the same copyright notice that appears on the sereen or printed page of such transmission. STOR is an independent not-for-profit organization dedicated to creating and preserving a digital archive of scholarly journals. For more information regarding JSTOR, please contact support @jstor.org, bupswww jstor.org/ Mon Ape 11 13:24:47 2005MULTIPLE SOURCES OF CHARACTER INFORMATION AND THE, PHYLOGENY OF HAWAIIAN DROSOPHILIDS RICHARD H. BAKER? AND Ros DESALLE "Department of Entomology, American Museum of Natural History, 79th Ste! at Central Bark West, [New York, New Yor 10024, USA; Ema olramninorg (RB) desalledumnlvorg (RD) "Department of Biology, Yee University, New Haxen, Connecticut 06520, USA eee dicho repr er Satria yaaa they amrmeroneae ean ti ge haere oy eterno rete Senha te cesemseearpcuaanmonsha eat RSI Ser ite ee cert nate casa tare crores fier cl geesdaie Sir the ieee erent Stim aceianineaccr nem Seaton Seay nga etre ce aay a Ene eee reninten aakrmemnrt metas Soe Sonepat ett seca ana sr a fnchapetaenransuarparntaepahmars idea cenmecs mgmisactove seamen Eee ees caereee Siam etn cee eee ae San ee elie seh ap ei outs Eycincan Remeenetlanemctes cecsdecearere Eetiare anaemia accra Brag sem eyeeteen ayo eemeree mame ae wee srs et ete co cs Baten sama ay a mpen spear Esmee The Hawaiian drosophilids include over 500 described species and have long been recognized as one of the most prominent examples of adaptive radiation and rapid ation in nature (Carson, 1987; Kane- shiro and Boake, 1987). Because of their re- markable diversity, these flies have been the subject of numerous systematic studies at various taxonomic levels (Spieth, 1966; Takada, 1966; Throckmorton, 1966; Car- son, 1970, 1982; Stalker, 1972; Hunt and Carson, 1983; Beverley and Wilson, 1985; DeSalle and Giddings, 1986; DeSalle etal, 1987; Grimaldi, 1990; DeSalle, 1992, 1995; Thomas and Hunt, 1993; Kambysellis et al, 1995). Despite this attention, many questions concerning the evolutionary re- lationships among Hawaiian drosophilids remain obscure. In the present study, we addressed a few of these areas by exam- ining the monophyly of the various mor- phological assemblages that have been es- tablished for this large group of flies and the relationships among these assem- blages. In particular, we addressed (1) whether the picture-winged Drosophila are ‘monophyletic and (2) which of the five ma- jor morphologically defined species groups (picture-winged, modified mouthparts, modified tarsi, white tip scutellum, and Antopocerus) are basal in the phylogeny. Overlain on these important organismal questions concerning Hawaiian flies are controversial issues of data analysis that also require attention, Picture-swinged Drosophila Monophyly Since Carson's (1970) seminal work on Hawaiian Drosophila systematics, the ques- ton of picture-winged monophyly has fe ‘mained a matter of debate, His chromosom- al phylogeny and the more recent yolk protein DNA sequencing study (Kambysellis et al, 1995) suggested that the three major 6541997 clades of picture-winged Drosophila (adiasto- la, grimshavi, planitibia) are monophyletic to the exclusion of flies in the group with mod- ified mouthparts. Two other molecular stud- ies, however, suggested that the adizstola subgroup is not closely related to the other two picture-winged groups. An immuno- precipitation study (Beverley and Wilson, {bes} placed the. group wi modified mouthparts as the closest relative ofthe plan- itibia subgrou subgroup clade. DeSalle et al. (1987) found additional sup- port for this relationship using mitochondri- al DNA sequence data. Basal Relationships of Species Groups Determining the most basal taxon or taxa in this group is important in light of comparative studies of ecologically inter- esting characters. Polarization of character states for ecological and life history traits, such as breeding substrate and ovarian morphology, is needed to complete schemes of character transformation and to infer the direction of evolution for these presumably adaptive features (Kambysel- lis et al,, 1995). Previous systematic work on Hawaiian Drosophila suggests that the ‘white-tip scutellum species group may be the most basal taxon, but this result re- ‘quires further attention. ‘The early morphological work of Takada (1966) and Throckmorton (1966) addressed the phylogenetic placement of a few of the Hawaiian Drosophila species groups. Al- though both studies (Figs. 1a, 1b) placed the white-tip scutellum flies as the most basal taxon, several of the other species groups were not included in the analyses. Spieth (1966) presented behavioral data that can be used as character state infor- ‘mation for taxa from a majority of the spe- cies groups. Because he did not produce a phylogeny for these flies, we analyzed his data using only those characters that were coded unambiguously (Spieth, 1966: table 1) and that are also phylogenetically infor- ‘ative. By this criterion, his original 30 be- havioral characters reduces to 13. We have chosen one representative from each of the five major species groups examined in our study to give an overall picture of the re- BAKER AND D:SALLE—MULTIPLE GENES AND HAWAIAN DROSOPHILA 655 lationships from behavioral characters. Figure Ic shows the topology of a tree based on the behavioral data and hypoth- esizes a basal position in the phylogeny for the white-tip scutellum flies. The most recent morphological treat- ment of these flies can be found in Gri- maldis (1990) monograph on the family Drosophilidae, for which he examined sev- eral representatives of Hawaiian drosophi- lids (Grimaldi, 1990: fig, 543; Fig, 1d). Al- though a white-tip scutellum fly emerged as one of the basal species in his consensus ‘ree, virtually none of the species groups are monophyletic. This result casts doubts ‘on whether any given species group can be designated as basal. Analytical Approaches Because we present sequence data from various gene sources, there are numerous process partitions (Bull et al,, 1993; Kluge and Wolf, 1993; Miyamoto and Fitch, 1995) that can be recognized within our data set. A controversy involving different philo- sophical approaches to phylogenetic hy- pothesis testing has led to many sugges- tions concerning how distinct process pattitions should be analyzed (for reviews Of this debate, see Kluge and Wolf, 1993; de Queiroz et al,, 1995; Miyomoto and Fitch, 1995; Hueselbeck et al, 1996; Nixon and Carpenter, 1996). The primary focus of this discussion concerns the suggestion that partitioning allows one to assess the equivalence of phylogenetic information derived from different sources and. that combining heterogeneous data violates certain assumptions of phylogenetic anal- ysis (Bull et al, 1993; de Queiroz, 1993) ‘This approach has been referred to as the prior agreement approach (Chippindale and Wiens, 1994). Some authors have even suggested that partitioning is a requisite to phylogenetic analysis and that the signals inherent in the various data matrices need to be considered separately (Miyamoto and Fitch, 1995) in a taxonomic congruence framework (Mickevich and Farris, 1981; Swofford, 1991). Alternatively, others have argued that data should be combined in all cases (Kluge, 1989; Barrett et al, 1991; Eer-656, SYSTEMATIC BIOLOGY vo. 46 var Pw Pw ag ‘HOGsca \ Yo ant occa ws / ws co o @) tb) = et = — ae — oa a wn neal es Hosera Tate rl HOGsca nrTs00 a ‘cogmul AiTads ©) @) ooo coon i Ficurs 1. Relationships proposed for Hawaiian Drosopile groups. MMTara = D. asistrichia; WTSbip = D. bipalita; MNtTach =D echyl, Atel Atclidrosphla; Nu = Nuldrsophile; MTSbas = D brsimacula; MTSper D. prissopada; MMTdis = D. dist; WTSfun ~ D. furgipends; PWGeng = D. engyochraen, PWGcru =D) ‘rucigea; MMTate = D. atrscutlata; PWPspe =D. specabiis; MMTco = ©. scolostomar PWAadl =D adistle; ‘ANTadu = D1 adunca (Antopceris) Species group abbreviations are as in Table I, (a) Redrawn fom Takada’s (2966) analysis of genitalia. (b) Redrawn from Throckmorton (1968) morphological analysis. (c) Taken from Spicth’s (1966) behavioral data. This toe is a single parsimony tree obtained by exhaustive search with PALP (Gteps = 22, consistency index = 0°58, retention index = 0.53). (4) Redrawn from Grimaldi’ (1990) cladistic Bnalysis of morphology.1997 Tans 1 Na space e Species group ee) “Aniopocers 8 Modified tarsus 150 Modified mouthpart 100 White-tip scutellam 100 Pictureswinged 100 plantiba 2 srinshawt 15 aisle 5 BAKER AND DrSALLE—MULTIPLE GENES AND HAWAIIAN DROSOPHILA 657 ‘The five species groups of Hawaiian Drosophila used in this study. Speces wed Abreitiont Banca ‘ANTadw tannic ANTian 1D dasenemis isdas D.petlopeza Mspet 1D minice MMfimim D scone MMTsoo Dig ‘wrsikt Disp WTSion D sestrs Pwr D eyrtoloma PwPeyr D disjuncta PWGdis D.linrosetae PwWGlin D astla Pwaadi Wie-ip satan iy curently being described by De Ken Kaneshiro nisse and Kluge, 1992; Kluge and Wolfe, 1993; Nixon and Carpenter, 1996) based on the rationale that the best explanation will include all of the relevant data and that a consensus approach implies arbitrary character weighting (Cracraft and Mindell, 1989). Much of this debate, however, has been conducted on theoretical grounds, s0 it is important to examine actual data sets to fully understand the implications of each approach. Our study includes data from eight gene regions and, therefore, of- fers a unique opportunity for this type of detailed examination. Materiats AND METHODS Flies Drosophilids from the Hawaiian Islands were collected in the wild by standard techniques, identified, and archived with voucher information by Dr. Kenneth Ka- neshiro. The flies slated for molecular anal- ysis were mailed alive to the American ‘Museum of Natural History and Yale Uni- versity, where upon receipt they were fro- zen at ~70°C. We attempted to use the same DNA from a single specimen as tem- plate for all DNA amplifications using PCR. When DNA from a particular indi- vidual was depleted, we used DNA from another individual of the same species col- lected at the same site. Exemplars Five major species groups were ana- lyzed in this study (Table 1). Atelidrosophila and Nudidrosophila (two very small genera of endemic Hawaiian flies) were not col- lected and so were not examined. At least two species from each of the five major species groups were analyzed for all of the genes in this study. One group that re- quired special attention, and therefore in- cluded more representatives, is the pic- ture-winged (PW) group. The PW group hhas 103 members, arranged by Carson (1970) into three major subgroupings; the planitibia subgroup (PWP),. the adiastola ‘subgroup (PWA), and the combined mono- phyletic subgroup of grimshawi, fascicula- selae, and havaiiensis (PWG). Previous im- munological data (Beverley and Wilson, 1985) and a. preliminary’ mitochondrial DNA (mtDNA) sequencing study (DeSalle et al, 1987) suggested that the adiastola subgroup is basal to all drosophilids from Hawaii. Carson's (1970) chromosomal data showed the PW flies as monophyletic with respect to one of his outgroups (D. mimica, ‘a member of the modified mouthpart spe- cies group). Outgroups Many of the genes used in this study are evolving rapidly; hence, the choice of out- ‘group is critical in establishing polarity of658, Tame 2 are provided inthe references listed SYSTEMATIC BIOLOGY vou. 46 Summary of the different gene regions used in this study. Primers for some of the gene regions Na dara Gone Atbreys Taal PP Refrene/ pine Mitochondrial mt 110196 168 :DNA 168 360 eae, 1992 NADH dehydrogenase 1 NDI 148 19S, 1992; Vogler et al, 1983, Cytochrome oxidase I COIIT 22958 Simon etal, 1994 Gytochrome oxidase II COI 36677 Brower, 1988 Nuclear uc 17301 ‘Alcohol dehydrogenase ADH 22258 Thomas and Hunt, 1995, ‘Acetycholinesterase ACHE 33 wo ;AGATCTGGARTCCCAA-3° Wingless ug 2 {CCACTCGRTACTGAAACGA-~ Hunchback ab 3 ow Ga abbas wal Ucughoul th paper ‘Numer af phylogenetically informative characters, relationships among these flies. DeSalle (1992) and: Thomas and Hunt (1993) sug- gested that the Hawaiian scaptomyzids Gubgenera Scaptomyza and Engiscaptomy- za) are the sister taxa to the Hawaiian dro- sophilids. Consequently, we chose a mem- ber of each of the two scaptomyzid subgenera as two of our outgroups: Scap- tomyza albovittata (HOGsca) and Engiseap- tomyza crassifemur (HOGcra). In addition, representatives of the two clades most closely related to all the Hawaiian taxa were also included. Based on DeSalleés (1992) study, these are the continental sub- geriera Drosophila and Sophophora. The spe- Ges chosen from these subgenera were D. ‘mulleri (Drosophila, COGmul) and D. mela- rogaster (Sophophora, COGmel). DNA Isolation and Manipulation DNA was isolated from single flies us- ing the small scale preparation outlined by DeSalle et al, (1993). A single fly usually yields enough template DNA for at least 20-30 PCR reactions using this approach. PCR primers designed for three mitochon drial gene regions, cytochrome oxidase Il, cytochrome oxidase Il, and 16S mitochon- drial ribosomal RNA plus NADH dehy- drogenase I, were used to compile the mi- tochondrial component of the data matrix. PCR primers for four unlinked nuclear gene regions, hunchback, wingless, acetyl- cholinesterase, and alcohol dehydrogenase, ‘were also designed and used to compile the nuclear gene region component of the data matrix. Information on the length of gene fragments and primer sequences is presented in Table 2, and accession nut bers for sequence data deposited GenBank are listed in Table 3. All sequence data for D. melanogaster and the ADH se- quences for D. mimica, Damulleri, D. adias- tola, D. crassifemur, D. silvestris, and S. al- bovittata were acquired from GenBank. For two of the nuclear genes, hb and wg, se- quence data were not obtained for a mid- dle portion of the PCR product: PER conditions difered forthe diferent rimer pairs, but in general all PCR reac- Eons were a variation on the general profile ‘of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, for a total of 35 cycles. Annealing temperature and number of cycles were usu- ally varied independently to optimize the conditions for each primer pait. Amplified DNA was sd_using one of three ‘methods: (1) direct sequencing of the dou- ble-stranded PCR product using manual di- deoxy sequencing and *5, (2) use of the ABI 373 automated sequencer to obtain sequenc- es using flourescent dideoxy terminator mix, and (3) use of the TA vector to clone the amplified product. Cloned products were1997 BAKER AND DsSALLE MULTIPLE GENES AND HAWAIAN DROSOPHILA 659 Tame 3. GenBank accession numbers for new Drosophila and Soaptomyze sequence data generated in study. Gees Species = “HE ADI __Coll___NDPe Dralustola U92986, U92007 SASS, U9iSS4 _UNR67 usin ven Dadunce —U92988, Us2999 94555, U9IESS Uni26s UtI94 Usd226 U9Rs1 UIIS1 94210 D creer 93000, U93001 uom68 usi7 voit Dieyrtloma’ —U93002, 093003. ‘U9d270_Us4i96 94228 U9i2A2 UBHO55 98212 Ddlisncta —U93004, Us3005 94561, U94562 9271 UBsI97 U9s229 U9NRAS UMS _U9I2I3 Dit 1U93006, 93007 94563, Uossed 098272 UssI98 99230 USK U94R57 Usi2I4 DD linensetae 093008, 093009 Uss565, Us4s65 94273 UssI99 Up4231 U9i2i5 U9458 U9s2i5 Dspe ‘Us3010, L93011 U94567, Usa56s USi274 Usi200 U94282 Uai246 U9259 U9i2I6 D.mimica —U93012, U93013 94569, U9NS7) 94275 vows usi217 mullen —U93014, U93015,_U9is71, U94S72 U94276 U9e234 UsI27 U0K26) L921 D petlopean —U93016, U93017 U9a573, U94S74 U9A277_U94R0S 94235 U9i24s 94261 U9I2I9 Stilwoitiata —U9B018, L93019 U94575, U94576. U9I278 Uem36 Use L9H262 U9i220 sitesi 95020, U93021 U94S77, U9E578 94279 U9e237 _Us4250 94263 L922 D.soonae —U98022, U93023. 94579, U945=) USL2RD U9405 U9i238 UMS U9s264 U9N222 D tanythric U93024, U93025 U9as81, U9N5R2 UBI28L U94207 94239 U9252 U9i26s 94223 1D dasenomia U9B026, U93027 _U94553, Usiss4 U9i2s2 U920s U9i240 UM253 U9266 U9I224 7 Speas wilh no accion ruber were ted by Thomas and Fant (993), ecepe Dinas (MST "Specks with no accession number were ltd by Desai (1982) “iuhtenpscuteom Ry urrenly beg described by Be. Ken Kaneshiro. then sequenced using the double-stranded ‘manual sequencing protocol with "S. Man- tual sequences were read into the program ‘MacVector and verified using this software. ‘Automated were transferred into MacClade and verified by visual inspection of the chromatographs produced from each "Thre aspects ofthe data collected in pre- vious Hawaiian ila studies prevent ‘us from combining, this information with ‘our molecular characters. In two studies (Takada, 1966; Throckmorton, 1966), the an- atomical data were not coded as character state information. For the other studies in which the data were coded as characters, there is either limited overlap of species be- ‘tween their studies and ours (Spieth, 1966; Grimaldi, 1990) or incomplete sampling of the various species groups (Carson, 1970; Kamibysellis et al, 1995). The third concerns the basic incompatibility of dis- tance data, in particular the i recip- itation data of Beverley and Wilson (1985), with character-based data. The inherent dif- ference in the nature of distance data and character-based data precludes the simulta- neous analysis and assessment incongru- ‘ence among these data (Brower et al, 1996). Data Analysis Alignment.—Mitochondrial_ ribosomal RNA ‘sequences were aligned using MA- LIGN (Wheeler and Gladstein, 1994), The alignments obtained were trivial because very few indels occur in the 165 sequences that we obtained, as previously reported by DeSalle (1992). Alignment of protein- coding regions was done by translating the DNA sequences into amino acid sequences and aligning the amino acid sequences in MEGALIGN (DNASTAR, version 1.02), us- ing the Clustal algorithm. For six of the protein-coding genes (wg, ACHE, ADH, COlll, COM, NDZ), these alignments were trivial because no more than a single ami no acid indel was hypothesized in any of these Clustal alignments. The nucleotide sequences of these six genes were aligned by codon to the amino acid alignments, and these aligned mucleotide sequences ‘were used in the data matrix. The seventh protein-coding gene (lib) has several hy- pervariable-length regions where amino acids are repeated multiple times, and it was not possible to obtain unambiguous alignment for these regions. Consequently, we modified the CULL (Gatesy etal, 1994) procedure to determine alignment-ambig-