446 SHORT COMMUNICATIONS and classification of birds. Yale Univ. Press, New parsimony*, Version 4.0d64. Sinauer, Sunderland, Haven, CT. MA. SIBLEY, C. G., AND B. L. MONROE. 1990. Distribution TARR, C. L., AND R. C. FLEISCHER. 1993. Mitochon- and taxonomy of birds of the world. Yale Univ. drial-DNA variation and evolutionary relation- Press, New Haven, CT ships in the amakihi complex. Auk 110:825-831. SICK, H. 1967. Rios e enchentes na AmazBnia coma VUILLEUMIER, E 1985. Forest birds of Patagonia: eco- obst&culo para a avifauna, p. 495-520. In H. Lent logical geography, speciation, endemism, and fau- [ed.], Atas do simp6sio sBbre a biota AmazBnica. nal history. Ornithol. Monogr. 36:255-304. Vol. 5 (Zoologia). Conselho National de Pesquis- WALLACE, A. k. 1852. On the monkeys of the Ama- as, Rio de Janeiro. zon. Proc. Zool. Sot. Lond. 20:107-110. SORENSEN,M. D. 1996. TreeRot. Computer program WALSH, S. I?, D. A. METZGER, AND R. HIGUCHI. 1991. distributed by the author. Univ. 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Songs of drial DNA sequences of 10 species of wrens in Trog- Northern and Southern House Wrens (T. aedon and T. lodytes and related genera suggest a new hypothesis of musculus, respectively) and Brown-throated Wrens (T. relationships for the group. The Winter Wren (T. trog- brunneicollis) have long trills, whereas Winter, Ru- lodytes) and the anomalous Timberline Wren (Thryor- fous-browed, Ochraceus (T. ochraceus), and Mountain chilus browni) are distantly related to the remainder of (T. solstitialis) Wrens all have longer, more varied Troglodytes. The latter group divides into a tropical songs largely lacking trills. montane group and a northern/lowland group that in- Our working hypothesis was that Winter Wrens cludes the northernmost two montane taxa (T. rufoci- might share a close phylogenetic relationship with the liatus, T. brunneicollis). Erection of the genus Nannus montane tropical forms of Troglodytes, representing an for the Winter Wren is proposed. Song evolution in the early lineage separate from the lowland forms. The complex has involved either convergent derivation or only previous phylogenetic study of the genus did not retention of primitive song types in distant lineages. include taxa critical to testing this hypothesis (Brum- field and Capparella 1996). Furthermore, morphomet- Key words: mtDNA sequences, Nannus, phyloge- ric studies of the entire genus by one of us (Escalona- ny, Troglodytes, wrens. Segura 1995) did not lead to firm conclusions regard- ing the evolutionary history of Troglodytes. For these North American ornithologists are well-acquainted reasons, we undertook a test of our hypothesis based with the relatively simple song of the House Wren on studies of mitochondrial DNA sequences. (Troglodytes aedon), in contrast to the long and com- plex song of the Winter Wren (T. troglodytes). Recent METHODS field work in the mountains of southern Mexico and Tissue samples are listed in Table 1. One or two rep- El Salvador brought two of us into contact with the resentatives of each mainland taxon that has at some Rufous-browed Wren (T. rufociliatus); we were struck point been considered as a species were included for by the extreme similarity of its song with that of Win- analysis, as well as the enigmatic Timberline Wren ter Wrens. Further examination of Troglodytes song (Thryorchilus browni, at times placed in Troglodytes) variation, in which two major song types were noted and outgroup taxa (White-breasted Wood-Wren Heni- corhina leucosticta, Pinyon Jay Gymnorhinus cyano- cephalus). Use of single or few individuals to represent ’ ’ Received 16 June 1998. Accepted 30 December taxa in phylogenetic analyses based on mitochondrial 1998. DNA sequence data follows Moore and DeFilippis SHORT COMMUNICATIONS 447 ,Tioglodytes aedon Nannus troglodytes T musculus Argentina s s T musculus Colombia 7: ochraceous B k 10 T brunneicollis T solstitialis Time FIGURE 1. Sonogramsfor representativeTroglodytes and Nunnus wren taxa examined in this study. (1997), based on low levels of within-taxon sequence lent resolution provided by this gene for species- and variation. generic-level questionsin other applications(Rice, un- Genomic DNA was extracted from each sample us- publ. data). This segmentwas amplified using conven- ing Qiamp tissueextraction kits available from Qiagen tional thermal-cycling techniques,with a thermal pro- (Valencia, California). A 534 base pair (bp) portion of file of denaturingat 95°C for 30 set, annealingat 55°C the ND2 gene was chosen for study, given the excel- for 30 set, and extension at 70°C for 90 set (Kocher 448 SHORT COMMUNICATIONS TABLE 1. Tissue samples used in this study. GenBank C~llCCtiOlP T,r\ur number access,on numher - Winter Wren Troglodytes troglodytes Illinois FMNH 1785 AF104975 Illinois FMNH 1778 AFI 04976 Northern House Wren T. aedon Illinois FMNH 1815 AF104979 Illinois FMNH 1783 AF104980 Southern House Wren T. musculus Oaxaca, Mexico MZFC uncat. AF104978 Brown-throated Wren T. hrunneicollis Oaxaca, Mexico MZFC 0MVP213 AFI 04973 Rufous-browed Wren T. rujixiliatus Chiapas, Mexico MZFC BMM607 AF104977 Ochraceous Wren T. ochraceus Cartago, Costa Rica LSU 19926 AF104982 Mountain Wren T. solstitialis Pasco, Peru LSU 8178 AF IO498 1 Tepui Wren T. rufulus Amazonas, LSU 7395 AF104983 Venezuela Timberline Wren Thryorchilus hrowni Cartago, Costa Rica LSU 19924 AF104974 White-breasted Woodwren Henicorhina leucosticta Lorcto, Peru KU 814 AF104972 Pinyon Jay Gymnorhinus Baja California, FMNH I667 AF104971 cyanocephalus Mexico a FMNH = Field Museum of Natural History, MZFC = Museo de Zoologia of Universidad Naciunal AuMnoma dc MBxico, LSU = Louriana State University Museum of Natural Science, KU = University of Kansas Natural History Museum. et al. 1989). Extension time was lengthened 4 set each peated for 25 cycles. Negative controls were used at cycle for 35 cycles. ND2 primers (H-6313: 5’. each step to test for reagent contamination. CTCTTATTTAAGGCTTTGAAGGC-3’ and L-5757: Numbers of variable and phylogenetically informa- 5’-GGCTGAATRGGMCTNAAYCARAC-3’) were tive molecular characters, as well as numbers and clas- developed by M. Sorenson (pers. comm.; H and L refer ses of transitions and transversions, were calculated to heavy and light strands, respectively, and numbers using MEGA 1 .Ol (Kumar et al. 1993). Rather than indicate relative position of primers on reference using percent sequence divergence as a distance mea- chicken sequence, Desjardins and Morais 1990). Am- sure to assess saturation, we used the Tamura-Nci Dis- plified product was purified on a low-melt (1%) tance, which considers percent base composition for NuSieve GTG agarose gel (FMC BioProducts; Rock- each individual, and is thus more robust than sequence land, Maine) electrophoresed for 4.5 min at 85-95 divergence measures (Kumar et al. 1993). All sequen- volts; bands containing target products were excised ces were deposited in GenBank (Table I). from the gel, and DNA recovered using Qiaquick spin Phylogenetic trees were estimated based on se- columns (Qiagen). Finally, purified product was am- quence data using the exhaustive search procedure of plified using one primer (heavy or light), and se- PAUP (version 3.1.1, Swofford 1991), an approach quenced with an ABI Prism Automated Sequencer guaranteed to identify shortest trees. Support for par- (Model 310). The thermal profile for both primer sys- ticular branches in resulting hypotheses was assessed tems was denaturing at 96°C for 10 set, annealing at using the branch-and-bound character bootstrapping 50°C for 5 set, and extension at 60°C for 4 min, re- algorithms in PAUP with 500 replica@ searches, and counts of unreversed synapomorphies. RESULTS BIOCHEMICAL PATTERNS Examination of sequences from the ND2 region re- vealed no insertions or deletions. Of the 534 base pairs examined, 2 15 were variable and 85 were phylogenet- ically informative. Partitioning bases by coding posi- tion revealed that 132 third position sites were vari- able, 60 of which were phylogenetically informative. At first and second positions, 61 (18 informative) and 22 (7 informative) sites, respectively, were variable. Saturation of sequence substitutions was not detect- ed either in the overall analysis or partitioning by cod- ing position (Fig. 2). However, given the small size of the data matrix, we conducted exploratory analyses FIGURE 2. Third-position saturation curves for ND2 with different transition:transversion weighting sequences, showing second-order polynomial fitted schemes (O:l, l:l, 2:1, 5: I, and 1O:l) and outgroup curves. Transitions are depicted with open circles, and taxa (Henicorhina leucosticta and Gymnorhinus cyan- transversions with open squares. ocephalus). These tests suggested that the topology de- SHORT COMMUNICATIONS 449 Henicorhina leucosticta 24 <50 8 Nannus troglodytes 22 Troglodytes brunneicollis 15 <50 1 T musculus 96 8 <50 2 c 2 T: aedon 9 i T rufociliatus <50 1 T: solstitialis 98 r T: ochraceus 0 T: rufulus 8 Thryorchilus browni 18 FIGIJRE 3. Cladogram showing the most parsimonious tree derived from analyses of sequences of the ND2 gene for montane Troglodytes wrens. Numbers above each node indicate bootstrap support, and those below each node are numbers of unreversed synapomorphics; those for terminal taxa represent numbers of autapo- morphic base pairs.