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Molecular Phylogenetics and EvolutionVol. 12, No. 3, August, pp. 273–281, 1999Article ID mpev.1999.0617, available online at http://www.idealibrary.com on

A Case of Rapid Diversification in the Neotropics: PhylogeneticRelationships among Cranioleuca Spinetails (Aves, Furnariidae)

Jaime Garcıa-Moreno,*,1 Peter Arctander,* and Jon Fjeldså†

*Zoological Institute, Department of Population Biology and †Zoological Museum, University of Copenhagen,Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark

Received March 23, 1998; revised September 24, 1998

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Relationships among the 18–19 species of spinetailsf the genus Cranioleuca are difficult to establish.ttempts based on traditional taxonomic charactersave failed because of a high degree of homoplasy.ost morphological characters vary independently,

roducing leap-frog patterns of variation along theastern Brazilian Andean track, and behavior andocalizations vary little. We use mtDNA sequenceata from the cyt b and ND2 genes in an attempto clarify relationships within the genus. We showi) that Cranioleuca represents a recent burst ofpeciation and (ii) that a set of species thoughty Maijer and Fjeldså (1997) to form a naturalroup is in fact a paraphyletic assemblage which alsoncludes humid forest species with different pig-

entations and vocalizations. However, synapomor-hic variation in the sequences is not sufficient tonambiguously resolve the relationships within theenus. Several species (C. baroni, C. antisiensis, C.yrrhophia, C. albiceps) show more than one haplo-ype, without any obvious correlation between geneticnd geographic or morphological variation, and theifferent species do not always show reciprocal mono-hyly in haplotype diversity. Nevertheless, low geneticifferentiation characterizes not only allopatric taxaut also some forms which are essentially sympatric,upporting species rank for the former. Our data sug-est a recent diversification and proliferation possiblyinked to Pleistocene climatic variation and its conse-uent vegetational shifts, at least in the Andeanpecies. r 1999 Academic Press

Key Words: Cranioleuca; mtDNA; speciation; biogeog-aphy; Andes.

1 To whom correspondence should be addressed at present address:useum of Zoology, University of Michigan, 1109 Geddes Avenue,nn Arbor, MI 48109-1079. Fax: (734) 763-4080. E-mail: jaimegm@

1mich.edu.

273

INTRODUCTION

Recent studies suggest a higher level of geneticifferentiation in some tropical avian species groupsGarcıa-Moreno et al., 1998; Roy, 1997; Hackett, 1996)han that found among birds in more temperate regionse.g., Klicka and Zink, 1997). However, because of themall number of studies it is difficult to draw generaliza-ions. We report here a case of a young radiation in theeotropics among birds of the genus Cranioleuca,hich show low levels of genetic differentiation relative

o other Neotropical genera.The genus Cranioleuca consists of 18–19 species

f spinetails (Ridgely and Tudor, 1994; Pachecot al., 1996; Maijer and Fjeldså, 1997) inhabitingost of South America (Fig. 1). The group is rather

omogeneous in vocalizations (Pacheco et al., 1996),ehavior, and appearance, being scansorial birdsressed mostly in buffy brown and grey, with rufousings and tail (Fjeldså and Krabbe, 1990; Ridgelynd Tudor, 1994). Several species replace each otherlong the Andes in rain-shadowed areas, but othersnhabit humid Andean slopes. Andean forms haveestricted distributions, whereas species inhabitingropical and subtropical lowlands are more widespreadFig. 1).

In his revision of the Furnariidae, Vaurie (1980) gavetrong attention to the color of the crown when clas-ifying the Cranioleuca species and allies. In theirescription of a new species, Maijer and Fjeldså (1997)uggested that a red cap is likely to be a plesiomorphicrait in this genus and therefore poorly suited forhylogenetic inference. They also pointed out thatther characters (e.g., vocalizations, ecology) do notorrelate well with coloration. Based on a number ofhared morphological characters and similar vocaliza-ions, they defined what they thought could be aatural group of species which replace each other alongn East Brazilian Andean track (defined by Silva,

995). The species in this group replace each other

1055-7903/99 $30.00Copyright r 1999 by Academic PressAll rights of reproduction in any form reserved.

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274 GARCIA-MORENO, ARCTANDER, AND FJELDSÅ

eographically, mainly in rain-shadowed areas withemi-humid forest and thickets: the morphologicallyery similar C. antisiensis in western Ecuador, C.enricae in mountain basins of northern Bolivia, and C.allida in eastern Brazil, are separated by birds withather different colors (C. baroni, C. albicapilla, C.yrrhophia, and C. obsoleta) (Fig. 1). Maijer and Fjeldsåuggested that, within this biogeographic track, thehylogenetic relationships would be correlated witheographic proximity rather than with morphologicalimilarity.Molecular data provide an independent set of charac-

ers from which robust phylogenies can be inferredAvise, 1994) and into which other characters (e.g.,orphology, vocalizations, etc) can be mapped. In this

aper we analyze DNA sequence data of spinetails ofhe genus Cranioleuca from two different mitochon-rial gene fragments, cytochrome b (cyt b) and NADHehydrogenase subunit II (ND2), to assess their phylo-

FIG. 1. Distribution of Cranioleuca spinetails. (a) Species considearenthesis not included in this study); (b) additional species includlotted on map (a). Capital letters refer to countries: (A) Ecuador, (B) Phe following areas: (1) Loja, (2) Cordillera Blanca and upper Huallag

enetic relationships. t

MATERIALS AND METHODS

amples

Tissues of 12 Cranioleuca species recognized byidgely and Tudor (1994) were obtained from the Bloodollection of the Zoological Museum of Copenhagen,

he Frozen Tissue Collections of the Academy of Natu-al Sciences of Philadelphia, and the Louisiana Stateniversity Museum of Natural Science. Included were

epresentatives of C. antisiensis (8), C. baroni (16), C.lbicapilla (3), C. albiceps (3), C. marcapatae, C. henri-ae (2) (Maijer and Fjeldså, 1997), C. pyrrhophia (8),. vulpina vulpecula (2), C. curtata, C. erythrops, C.bsoleta, and C. demissa (Appendix 1). Samples of C.ulphuriphera, C. pallida, C. semicinerea, C. hellmay-ii, C. gutturata, and C. muelleri were not available fornalysis. Hellmayrea gularis, the closest taxon to Cra-ioleuca and Certhiaxis (Vaurie, 1980), was chosen asutgroup. Additional more distant outgroups were As-

by Maijer and Fjeldså (1997) to form a monophyletic group (those inin the present study. Important localities mentioned in the text are, (C) Bolivia, (D) Uruguay. Numbers adjacent to birds’ ranges refer to

alley, (3) Lima, (4) La Paz, and (5) Cochabamba.

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275RAPID DIVERSIFICATION IN SPINETAILS

uscus. Published sequences of Asthenes (Edwards etl., 1991) and Lepidocolaptes (Garcıa-Moreno and Silva,997) were complemented with sequences obtained forhis work. Details about the origin of the samples areummarized in Appendix 1.

NA Extraction

DNA was extracted from blood samples preserved inDTA (Arctander, 1988) or in DMSO (Arctander andjeldså, 1994). Samples were washed in blood buffer

NaCl 0.1 M, Tris–HCl 0.01 M, pH 8, EDTA 0.001 M, pH), incubated in CTAB buffer (Doyle and Doyle, 1987) at5°C, and extracted with chloroform:isopentyl alcohol24:1 v:v). The DNA was then precipitated with isopro-anol, cleaned twice with ice-cold ethanol, and finallyissolved in TE buffer (10 mM Tris–HCl, pH 7.6, 1 mMDTA).

CR and Sequencing

PCR products were obtained using primer pairs5215 and H5578 (Hackett, 1996) for ND2, as well as14841 and H15149 (Kocher et al., 1989) and L15564nd H15915 (Edwards et al., 1991) for cyt b. Double-tranded PCR was performed in 20-µl reaction volumessing equal amounts of primers. Five microliters of theeaction was electrophoresed in a low-melting-pointgarose gel; the bands were cut out and melted in 400l TE buffer. One microliter of this solution was used asemplate to generate single-stranded DNA by asymmet-ic PCR amplification. The conditions were identical tohose of the balanced-primer reaction except that theeaction volume was increased to 50 µl, the limitingrimer was diluted a hundredfold, and the number ofycles was increased by three extra cycles. Reactionroducts were cleaned by washing with ddH2O throughltrafree-MCf (Millipore Corp.) 30,000 NMWL filtersnd concentrated to a final volume of 18 µl, of which 8l was used for DNA sequencing.An alternative for generating single-stranded PCR

roducts was to produce double-stranded PCR productssing one biotynilated primer in 35-µl reactions. Theroducts were purified using Dynal magnetic beadsollowing the protocol of the supplier.

Dideoxy sequencing was performed following therotocol for Sequenase v. 2.0 (United States Biochemi-als). The products, labeled with S35, were resolved in% acrylamide gels. Sequences have been submitted toenBank under Accession nos. AF053773–AF053822nd AF053825.

nalyses

Sequences were aligned by eye using the programseqApp 1.9 (Gilbert, 1992). Because the fragmentsequenced correspond to protein coding genes, transla-ion to amino acid sequence was verified using Mac-

lade 3.06 (Maddison and Maddison, 1992). To calcu- o

ate the number of substitutions (total, transitions,ransversions) between homologous DNA sequences,e used the program CS3 (H. Siegismund, unpub-

ished). These values were critically assessed because its known that paralogous mitochondrial and nuclearequences exhibit differences in their substitution pat-erns (Arctander, 1995). Maximum likelihood analysesere performed using the program DNAml in Phylip

version 3.57; Felsenstein, 1996) using a transition/ransversion ratio of 23 (found empirically) and threeifferent substitution rate categories (which do notecessarily correspond to codon positions) with rates of.25, 1, and 3, and frequencies of 0.7, 0.25, and 0.05,espectively. These values were chosen to crudely emu-ate a gamma distribution with a parameter of 0.5Yang, 1996). Parsimony analyses were carried outsing the branch and bound search option in PAUP.1.1 (Swofford, 1991) followed by 500 bootstrap repli-ates with random addition sequence. Neighbor-joiningnalyses were performed using MEGA 1.01 (Kumar etl., 1993).

RESULTS

evels and Patterns of Variation

A total of 445 bp corresponding to two fragments ofhe mitochondrial cyt b gene were sequenced for 12ranioleuca species and three outgroups included in

his study. There were 58 variable sites detected amongpecies in the Cranioleuca genus, 30 of which werenformative regarding the relationships among them.f all substitutions within this group, 96% were transi-

ions (ts) and the remaining 4% transversions (tv).ifteen percent of the substitutions occurred at firstodon position, none at second codon position, and 85%t third codon position, a pattern similar to that foundn other studies of closely related species.

Similarly, a total of 312 bp were sequenced for ND2.f these, 33 positions were variable within the Cranio-

euca group, and only 14 positions were informativeegarding the relationships within the genus. Of allubstitutions within this group, 98% were transitionsnd the remaining 2% transversions. Seventeen per-ent of the substitutions occurred at first codon posi-ion, 12% at second codon position, and 71% at thirdodon position. The levels and patterns of variationxhibited by the sequences are summarized in Table 1.

ntraspecific Variation

To assess the level of intraspecific genetic variation,e compared a minimum of 240 bp from one of the cyt b

ragments (the 38 end) for all species of which we hadeveral samples (Appendix 1). Five out of six samples of. antisiensis shared an identical haplotype. The sixth

ne differed from the others by two ts. The different

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276 GARCIA-MORENO, ARCTANDER, AND FJELDSÅ

aplotypes originated from samples coming from theame locality (Tierra Colorada in Loja, Ecuador).Two different haplotypes were also detected among

6 samples of C. baroni, differing by a single ts. Allamples of the subspecies C. b. zaratensis from Limahared the same haplotype, but this was also present inamples of other subspecies (C. b. baroni and C. b.apitalis, which are almost twice as big as zaratensis).s in C. antisiensis, both haplotypes were detected inamples coming from the same locality (Quebradaucavado in Cordillera Blanca, Peru).Among the eight samples of C. pyrrhophia, two

aplotypes were also detected differing from each othery four ts. One of the haplotypes is identical to C.bsoleta’s (for this fragment) and differs by only one tsrom C. henricae’s. No clear geographic segregation ofhe haplotypes was seen, since one of the haplotypesas shared by samples coming from Uruguay andhuquisaca and Potosı in Bolivia, while the other wasresent in individuals from Chuquisaca and Co-habamba.Among other species for which more than one sampleas available, C. vulpina (2), C. henricae (2), C. albica-illa (3), and C. albiceps (3), we detected differencesnly in the last one, with haplotypes differing by aingle transition.

hylogenetic Analysis

Due to the small number of informative sites in eachene region, the 445 bp of cytochrome b and the 312 bpf ND2 were pooled together in order to try to resolvehe phylogenetic relationships among Cranioleuca spe-ies. Figure 2 depicts the topologies of the best trees

TAB

Uncorrected Pairwise Sequence Divergence (abovSubstitutions (below Diagonal) for 757 bp of mtD

1 2 3 4 5 6

1 C. baroni a — 0.004 0.010 0.012 0.023 0.026 0.2 C. baroni b 3/0 — 0.010 0.013 0.024 0.026 0.3 C. antisiensis 7/0 7/0 — 0.005 0.018 0.024 0.4 C. curtata 9/0 10/0 4/0 — 0.021 0.027 0.5 C. erythrops 16/1 17/1 12/1 15/1 — 0.020 0.6 C. demissa 19/0 19/0 18/0 20/0 14/1 — 0.7 C. albicapilla 26/1 25/1 24/1 27/1 17/2 23/18 C. pyrrhophia a 25/2 24/2 21/2 26/2 18/3 21/2 199 C. pyrrhophia b 25/1 24/1 21/1 24/1 18/2 21/1 190 C. obsoleta 28/1 29/1 25/1 27/1 21/2 25/1 241 C. henricae 22/1 23/1 19/1 21/1 15/2 20/1 182 C. marcapatae 27/1 27/1 30/1 32/1 25/2 29/1 303 C. albiceps 28/1 27/1 30/1 33/1 28/2 28/1 324 C. vulpina 28/2 27/2 30/2 33/2 25/3 23/2 265 Hellmayrea gularis 61/8 62/8 58/8 62/8 64/9 64/8 666 Asthenes dorbignyi 77/12 76/12 76/12 80/12 79/13 76/11 757 Lepidocolaptes fuscus 64/39 63/39 63/37 64/39 67/40 66/36 68

roduced by (a) the maximum likelihood considering t

ifferent rates of nucleotide substitution and (b) theeighbor-joining algorithm based on uncorrected dis-ances.

The branching order of the maximum likelihood treeFig. 2a) was not affected by different ts:tv ratios (5, 13,3) or gamma parameters (equal rates, 0.1, 0.5), and itas also recovered among the 15 most parsimonious

rees (2Ln: 2425; length: 306 steps).The other 14 equally parsimonious trees (length:

06) shared the overall topology with the tree shown inig. 2a, with four sources of dissimilarity: (a) C. baroniand C. baroni b were either clustered together in a

lade or C. baroni b is shown basal to C. baroni a; (b) thelade comprising C. antisiensis and C. curtata is sistero C. baroni, or C. baroni is basal to it; (c) the position of. demissa and C. erythrops varied, as their relativeositions were interchangeable; and (d) the position of. albicapilla, the pyrrhophia/obsoleta/henricae clade,nd the albiceps/marcapatae/vulpina clade varied,ith all three possible arrangements (C. albicapillaost basal, sister to pyrrhophia clade, and sister to

lbiceps clade).The trees obtained with distance-based algorithms

Fig 2b; 2Ln 2431; length 308) also shared the overallopology with the maximum-likelihood tree. They dif-ered from it as the group made of C. albiceps–C.arcapatae was the most basal branch within Cranio-

euca, with C. vulpina forming the next branch.A strict consensus of the branching orders obtainedith different algorithms yielded an unresolved comb-

ike arrangement within Cranioleuca, with C. marcapa-ae and C. albiceps, C. antisiensis and C. curtata, and

1

iagonal) and Numbers of Transition/TransversionA among Cranioleuca Spinetails and Outgroups

8 9 10 11 12 13 14 15 16 17

0.036 0.034 0.038 0.030 0.038 0.038 0.040 0.091 0.118 0.1420.034 0.033 0.040 0.032 0.038 0.037 0.039 0.093 0.116 0.1410.031 0.030 0.035 0.027 0.042 0.042 0.044 0.090 0.120 0.1420.037 0.033 0.037 0.029 0.045 0.045 0.047 0.093 0.122 0.1420.028 0.027 0.031 0.023 0.037 0.040 0.037 0.097 0.122 0.1480.031 0.030 0.035 0.029 0.041 0.039 0.034 0.098 0.119 0.1450.026 0.025 0.032 0.024 0.041 0.042 0.036 0.099 0.116 0.149

— 0.012 0.021 0.015 0.042 0.045 0.032 0.102 0.120 0.1458/1 — 0.012 0.008 0.041 0.044 0.033 0.097 0.114 0.143

15/1 9/0 — 0.009 0.043 0.050 0.040 0.101 0.121 0.14910/1 6/0 7/0 — 0.037 0.042 0.033 0.097 0.114 0.14430/1 30/0 32/0 27/0 — 0.016 0.039 0.106 0.107 0.14733/1 33/0 38/0 32/0 12/0 — 0.041 0.114 0.106 0.14322/2 24/1 29/1 24/1 28/1 30/1 — 0.108 0.111 0.14567/10 64/9 67/9 64/9 69/9 77/9 71/10 — 0.111 0.13577/14 73/13 78/13 73/13 66/13 67/13 70/13 74/10 — 0.13164/41 64/40 68/40 64/40 66/38 64/40 64/40 65/33 62/33 —

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277RAPID DIVERSIFICATION IN SPINETAILS

ies enforcing monophyly on Maijer and Fjeldså’s (1997)roposed group (C. antisiensis, C. baroni, C. albica-illa, C. pyrrhophia, C. henricae, and C. obsoleta) withespect to other Cranioleuca species (2Ln: 2646), or aorthern Andean versus southern species dichotomy

2Ln: 2498), were rejected by the test of Kishino andasegawa (1989) (P , 0.001). None of the optimal

opologies recovered by the different phylogenetic analy-es were significantly different from each other accord-ng to the same test. In all topologies Cranioleuca washown as a monophyletic group, with H. gularis as itslosest relative and Asthenes more distant.

berrant Sequences

Amplification of three samples of line-cheeked spin-tail (C. antisiensis) from Loja, Ecuador, consistentlyielded a cyt b sequence different from that of otheramples of the same species. This sequence showed aivergence of 10.6–12% (39–44 ts; 7–8 tv) comparedith the other Cranioleuca cyt b sequences, includingther samples of C. antisiensis (11.2%). No stop codonsere found in the sequence, and only a single aminocid substitution was found compared to other Cranio-euca (Serine AGC for Glycine GGC, base change at

FIG. 2. Two hypothesis about the phylogenetic relationships amoifferent rates of nucleotide substitution (2Ln 2425; length 306). Thranches are drawn proportionately to the amount of change along thequences (branch length not to scale); (b) Neighbor-joining tree baseds suboptimal in parsimony and maximum likelihood analyses, it hasnresolved despite a high bootstrap value grouping it to the southerercentage bootstrap values higher than 50% after 500 replicates (un

osition 15910 of the chicken mtDNA; Desjardins and t

orais, 1990). A phylogenetic analysis placed thisequence outside the Cranioleuca cluster and evenasal to H. gularis, closer to the sequence of Asthenes ashown in Fig. 2a. It is beyond the scope of this work toetermine whether these sequences are nuclear homo-ogues of cyt b or pertain to a ‘cryptic’ taxon within theenus.

DISCUSSION

Although the potential amount of informative charac-ers from sequence data is immense, molecular studiesave failed to resolve the phylogeny of some rapidlypeciating groups. Cytochrome b and ND2 have repeat-dly been used to investigate the relationships oflosely related species (e.g., Hackett, 1996; Roy, 1997;arcıa-Moreno et al., 1998, for some recent tropicalvian examples; reviewed by Avise, 1994). In this study,equences of over 750 bp of mtDNA were not sufficiento confidently resolve the relationships among spin-tails of the genus Cranioleuca.Cues suggesting that Cranioleuca represent a recent

peciation burst are:(a) Monophyly. Analysis of mtDNA sequences suggest

Cranioleuca spinetails. (a) Maximum likelihood tree accounting forme tree topology was found among the 15 most parsimonious trees.

. The stippled line indicates the position of the aberrant C. antisiensisuncorrected distances (2Ln 2431; length 308). Although the topologyetter agreement with nonmolecular data. C. albicapilla is shown aslade (see text for explanation). Figures above the branches indicateparsimony criterium in (a) and neighbor-joining in (b)).

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hat these spinetails form a monophyletic group com-

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278 GARCIA-MORENO, ARCTANDER, AND FJELDSÅ

ared to other furnarids. This result was always ob-ained, regardless of the method used for reconstruct-ng the phylogeny or the rooting taxon(s) used. Basedn data from morphology, vocalizations, and proteinlectrophoresis, Braun and Parker (1985) suggestedhat Hellmayrea is closer to Cranioleuca than to Synal-axis, where it was placed by others (e.g., Meyer dechauensee, 1966; Vaurie, 1980). Our analyses indicatehat H. gularis is closer to Cranioleuca than Astheness, although clearly distinct from them.

(b) Low intrageneric genetic variation. Braun andarker (1985) concluded that the average genetic dis-ance between Cranioleuca species is similar to thatound among other congeneric passerine species. Simi-arly, the range of interspecific mitochondrial sequenceivergence in Cranioleuca (0.5–4.7%, mean 3.1%; Table) is within the range found in other intragenericomparisons (e.g., 1.4–8.2%, Krajewski and Fetzner,994; 2.3–8.2%, Zink, 1996; 0.1–10.2%, Cicero andohnson, 1995), despite the fact that 14 different haplo-ypes were compared. Even though we detected vari-ble sites among the sequences (33 ND2—11%; 54 cyt—12%), these appear few compared with other generae.g., 28% in cyt b in Puffinus, Austin, 1996; 28.5% in cytand ND2 in Ramphocelus, Hackett, 1996). Moreover,

ery few of the variable sites were actually informativeoncerning the relationships among Cranioleuca (14D2—4.5%; 30 cyt b—6.7%) and they were low com-ared to others (Puffinus 17.3%; Ramphocelus 11.8%).The Cranioleuca species are also closely related in

omparison to some other speciose genera of tropicalirds (Hackett, 1996; Roy, 1997; Garcıa-Moreno et al.,998; Garcıa-Moreno and Fjeldså, 1999). Within thendean Metallura hummingbirds, Atlapetes brush-nches, and the most recently derived Ochthoeca chat-yrants, species divergence data ranged from 0.2 to.5% (average 5.3% 6 2.2), 0.3 to 8.0% (average.1% 6 1.9), and 1.7 to 6.7% (average 5.1% 6 1.6),espectively.(c) Lineage sorting not fully completed. Analyses oftDNA sequences from recently split taxa can result in

rees that do not reflect species limits. If the rate ofsolation of populations is higher than the mutationnd fixation rates in given lineages (coalescence time,hich is expected to be Ne/2 generations for mtDNA;vise, 1994; Moore, 1995), then the gene trees may notarallel the evolution of the species (Stepien and Kocher,997). Thus, haplotypes in one species fail to showeciprocal monophyly and can be more closely related toaplotypes in the sister species than to those in theirwn (Klicka and Zink, 1997).Within our data set, we detected more than one cyt b

aplotype in four of the species for which more than oneample was available (C. albiceps, C. antisiensis, C.aroni, and C. pyrrhophia). There is at least one case

hat could exemplify ancestral polymorphisms being s

arried through the speciation event(s) and randomlyxed in the descendant populations (Stepien and Kocher,997). Within the Southern clade (Fig. 2), Belton (inidgely and Tudor, 1994) suggested that C. obsoleta isnly a subspecies of C. pyrrhophia. C. obsoleta actuallyesembles a juvenile C. pyrrhophia, and the two speciesntergrade in the contact zone in southern Brazil andruguay, where all individuals show intermediate plum-ges (L. Joseph, pers. comm.). In our analysis the twoaplotypes of C. pyrrhophia do not form a separate andistinct group, and one of the C. pyrrhophia haplotypess actually more similar to those of C. obsoleta and C.enricae than to the other haplotype of its own species

Fig. 2, Table 1).(d) Lack of correlation between morphological and

enetic variation. Our results show that morphologicalariation within species was likewise uncorrelatedith mitochondrial genetic variation. This is obvious in

nterspecific comparisons (e.g., a 2.7% sequence diver-ence contrasting a very high phenotypic similarityetween C. henricae and antisiensis; and genetic simi-arity contrasting phenotypic dissimilarity between C.enricae and pyrrhophia and between C. antisiensisnd curtata). It is also illustrated within C. baroni, inhich there is tremendous variation in size. Birds from

he upper Huallaga valley or Cordillera Blanca weighearly twice as much as birds living at the samelevation in Lima, even though there is no definitivearrier between these sites (Maijer and Fjeldså, 1997).e detected two different haplotypes differing by one

ransition in 240 bp (but we did not sequence all 16ndividuals for the full 757 bp score). All small-sizedndividuals (which are considered a distinctive subspe-ies, C. b. zaratensis) shared the same haplotype, buthis was also present in individuals of other subspecies.

This result is not surprising since a single-characterorphological change can occur much more rapidly

han the time it takes to accumulate mutations ran-omly. We detected two different haplotypes differingy one ts among three individuals of C. albiceps; yet thewo samples that shared the same haplotype differedorphologically as one individual collected in Cocapata

northern Cochabamba) was white capped, like birdsrom La Paz. The Cocapata population showed a com-lete integradation between the northern white-cappednd the southern orange-capped subspecies albicepsnd discolor. Remsen (1984) reported a case in Cranio-euca albiceps in which the color of the crown cap hadhanged in one locality over 50 years (see Fjeldså, 1983,nd Losos et al., 1997, for other examples of very rapidorphological change). He suggested that the differen-

iation (north to south) of white-capped C. marcapataeeskei, rufous-capped C. m. marcapatae, white-capped. a. albiceps, and orange-capped C. albiceps discoloray represent differential local fixation of alleles in a

ingle species.

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279RAPID DIVERSIFICATION IN SPINETAILS

The lack of signs of intergradation between currentlyccepted species (except for C. pyrrhophia and obso-eta), including the cases of marginal sympatry inhich some of them have been recorded on the same

lope (C. albiceps/henricae, C. albicapilla/baroni, C.lbicapilla/marcapatae, and possibly C. antisiensis/rythrops and C. henricae/pyrrhophia), suggest speciesanks rather than variable fixation of characters be-ween populations of only a few species. The currentesearch on parapatric and marginally sympatric birdaxa is dominated by demonstration of limited inter-reeding and replacement where species meet (Gill,998).

iming and Patterns of Phylogenetic Eventsin Cranioleuca Spinetails

The evidence from our study indicates a recentifferentiation in Cranioleuca spinetails. However, be-ause of incomplete resolution with the data available,t is not possible to discriminate between alternativeistories (notice the very short lengths of certainranches within Cranioleuca in Fig. 2). In any case, thehylogenies reflect the existence of several groups ofranioleuca spinetails, suggesting that Maijer andjeldså’s East BrazilianAndean assemblage is paraphy-

etic and its morphological characteristics are plesiomor-hies within the genus. We think that the molecularhylogeny depicted in Fig. 2b could best describe theistory of the group since it agrees best with otherharacters (e.g., nest structure) and it has the highestupport–conflict ratio (data available from first authorn request).The largest group within the phylogeny encompasses

he majority of Cranioleuca included in the study andan be further subdivided into two subunits: (i) mon-ane birds in northern South America and (ii) mostlyowland species mainly in the south and east of theontinent (Figs. 1 and 2). The northern group compriseshe birds living in humid submontane forests (C. ery-hrops, C. curtata, C. demissa), with C. antisiensis and. baroni in somewhat drier zones. It could also include. albicapilla (of intermontane, semihumid forest),lthough we consider its position as unresolved (Fig. 2).ll the birds included in this group live in the moun-

ains and build pendant nests (Vaurie, 1980). Theecond subgroup includes C. pyrrhophia, C. henricae,nd C. obsoleta. All these species have nests supportedrom the bottom, prefer woodlands and dry forests, andnhabit mainly lowlands (although C. pyrrhophia cane found at up to 3000 m in Bolivia and C. henricaenhabits isolated pockets of dry forest in northernolivia).C. marcapatae and C. albiceps make up a second

roup of species. These have a distinctive vocal reper-oire compared to other species (Maijer and Fjeldså,

997), and their pendant nests and association with c

umid montane habitats could reflect the ancient condi-ions of the group (note that H. gularis is also associ-ted with humid forests). Finally, C. vulpina has aroadAmazonian distribution, where it inhabits species-oor early successional stages on river islands and riverdges, and its vocalizations are somewhat differentrom those of other Cranioleuca spinetails (Pacheco etl., 1996; N. Krabbe, pers. comm.).In order to provide a time frame (with a very large

rror; see Hillis et al., 1996), we assumed that ND2 andyt b are evolving at roughly the same rate as the wholeitochondrial genome in other birds (2% substitutions

er million years; see Klicka and Zink, 1997; but seeoewe and Scherer, 1997, and Gibbons, 1998). This sets

he Cranioleuca radiation within the last 2 millionears. If we consider a rate proposed for cyt b by Irwint al. (1991) of 10% third position substitutions perillion years, it indicates an even more recent radia-

ion: the average of third position substitutions amongranioleuca species is 7.7% (range 0.8–11.9%), suggest-

ng a radiation within the last million years. In thisespect, the genus conforms with other genera of as-umed Pleistocene origin (Klicka and Zink, 1997).A model of rapid divergence of relict populations (see

aterson, 1985; Clarke and Walter, 1995) applies wello areas with complex topography (Vrba, 1992). Itroposes the interaction of ecoclimatic changes, whichave been more drastic during the past 800,000 years

Hooghiemstra et al., 1993), and topographic relief ashe driving force behind diversification (see Roy et al.,997). Despite some evidence of Pleistocene vegeta-ional changes in the lowland floodplains (van derammen and Absy, 1994; Colinvaux et al., 1996) it isnclear whether such changes caused speciation withinhese plains (Vrba, 1992; Fjeldså, 1994). Instead, phylo-enetic data and the morphological distinctness ofome lowland spinetails suggest that there could haveeen independent adaptive shifts leading to coloniza-ions of lowland plains. This needs to be evaluated byncluding all species of Cranioleuca and by studies ofther large genera (e.g., Synallaxis) inhabiting low-ands as well as montane areas. A more thoroughiscussion of possible speciation scenarios would re-uire a much finer scale sampling than the one avail-ble for this study.

ACKNOWLEDGMENTS

This work was part of a Ph.D. project by the first author, supportedy the Danish Natural History Research Council (Grant no. 11-0390).his 5-year grant to the Centre for Tropical Biodiversity providedxcellent working conditions for the second and third authors. J.irungi, R. Bleiweiss, A. Helbig, and three anonymous reviewers

ommented on previous versions of the manuscript.

A

A

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280 GARCIA-MORENO, ARCTANDER, AND FJELDSÅ

REFERENCES

rctander, P. (1988). Comparative studies of avian DNA by restric-tion fragment length polymorphism analysis: Convenient proce-dures based on blood samples from live birds. J. Ornithol. 129:205–216.

rctander, P. (1995). Comparison of a mitochondrial gene and acorresponding nuclear pseudogene. Proc. R. Soc. Lond. B 262:13–19.

rctander, P., and Fjeldså, J. (1994). Andean tapaculos of the genusScytalopus (Aves, Rhinocryptidae): A study of speciation usingDNA sequence data. In ‘‘Conservation Genetics’’ (V. Loeschke, J.Tomiuk, and S. K. Jain, Eds.), pp. 205–225. Birkhauser, Basel.ustin, J. J. (1996). Molecular phylogenetics of Puffinus shearwaters:Preliminary evidence from mitochondrial cytochrome b gene se-quences. Mol. Phylogenet. Evol. 6: 77–88.

vise, J. C. (1994). ‘‘Molecular Markers, Natural History and Evolu-tion,’’ Chapman & Hall, London.

APPE

Collecting Localities for the T

Ser. no. Species Country

248 C. albicapilla Peru2591001243, S244 C. antisiensis Ecuador245265, S2681516263, S264, S267 f C. antisiensis Ecuador270 C. baroni Peru271–S277269278–283285297b C. curtata Ecuador-7421c C. demissa VenezuelaK1 C. erythrops Ecuador1268, S1269 C. henricae Bolivia1466 C. marcapatae Peru2456, S2457 C. albiceps Bolivia2422AT8 C. obsoleta Paraguay287 C. pyrrhophia Bolivia16921702, S1703, S17111724J153, LJ154 Uruguay1197, 4729b C. vulpina Ecuador306 Hellmayrea gularis Ecuador136 Asthenes arequipae Peru151 A. dorbignyid BoliviaA 18 Lepidocolaptes fuscuse Brazil

a Samples from the Blood and Tissue collection of the Zoological Mub Sample from the National Academy of Sciences, Philadelphia.c Sample from Louisiana State University Museum of Natural Sciend Sequenced only ND2 for this work; cyt b sequence was taken frome Sequenced only the last 250 bp of cyt b (38 end) for this work. Sequ

ilva (1997).f Samples that amplified aberrant sequences.

raun, M. J., and Parker, T. A. (1985). Molecular, morphological, and

behavioral evidence concerning the taxonomic relationships of‘‘Synallaxis’’ gularis and other Synalaxines. Ornithol. Monogr. 36:333–346.icero, C., and Johnson, N. K. (1995). Speciation in sapsuckers(Sphyrapicus): III. Mitochondrial-DNA sequence divergence at thecytochrome-b locus. Auk 112: 547–563.

larke, A. R., and Walter, G. H. (1995). ‘‘Strains’’ and the classicalbiological control of insect pests. Can. J. Zool. 73: 1777–1790.olinvaux, P. A., de Oliveira, P. E., Moreno, J. E., Miller, M. C., andBush, M. B. (1996). A long pollen record from lowland Amazonia:Forest and cooling in glacial times. Science 274: 85–88.esjardins, P., and Morais, R. (1990). Sequence and gene organiza-tion of the chicken mitochondrial genome. A novel gene order inhigher vertebrates. J. Mol. Biol. 212: 599–634.oyle, J. J., and Doyle, J. L. (1987). A rapid DNA isolation procedurefor small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11–15.

dwards, S. V., Arctander, P., and Wilson, A. C. (1991). Mitochondrialresolution of a deep branch in the genealogical tree for perching

IX 1

sues Included in this Studya

District Locality

Apurimac Bosque Ampay 3400 mCuzco Chainapuerto

Calca 4150 mLoja Tierra Colorada 1700 m

Utuana 2550 mCelica

Azuay St. Isabel to Giron 1750 mLoja CelicaPasco PariamarcaLima Pueblo Chichas, OyonAncash Matara in Q. Ulta

Quebrada PucavadoRurichinchay

Zamora-Chinchipe Cord. del Condor, 1425 mAmazonas Cerro de la Neblina

TanahuagaLa Paz Inquisivi, 2350 mCuzco Pillahuata 2800 mCochabamba Pujyani, Cocapata

CocapataCanindeyu P. N. San RafaelCochabamba 10 km SW AiquileChuquisaca Palmarcito 18.35S/64.52W

SopachuyPotosı Parinolqui PampaRio Negro El Abrojal, ca 45 km NE of Fray BentosNapo Rio Napo/Aguarico, 150 mLoja CajanumaArequipa Cerro QuishuarniyocCochabamba Cerro Khenwa-SandoraSao Paulo Sao Luis do Paraitinga

m, Copenhagen unless stated otherwise.

.wards et al. (1991).es for ND2 and the b1–b2 fragment of cyt b from Garcıa-Moreno and

ND

is

seu

ceEdenc

birds. Proc. R. Soc. Lond. B 243: 99–107.

F

F

F

F

G

G

G

G

G

GH

H

H

I

K

K

K

K

K

L

L

M

M

M

M

P

P

R

R

R

R

S

S

S

V

V

V

Y

Z

281RAPID DIVERSIFICATION IN SPINETAILS

elsenstein, J. (1993). PHYLIP (Phylogeny Inference Package). Ver-sion 3.5. Dpt. of Genetics, University of Washington, Seattle.

jeldså, J. (1983). Ecological character displacement and characterrelease in grebes (Podicipedidae). Ibis 125: 463–481.

jeldså, J. (1994). Geographical patterns for relict and young speciesof birds in Africa and South America and implications for conserva-tion priorities. Biodiversity Conserv. 3: 207–226.

jeldså, J., and Krabbe, N. (1990). ‘‘Birds of the High Andes,’’Zoological Museum Copenhagen and Apollo Books, Svendborg,Denmark.arcıa-Moreno, J., and Silva, J. M. C. (1997). An interplay betweenforest and non-forest South American avifaunas suggested by aphylogeny of Lepidocolaptes woodcreepers (Dendrocolaptinae). Stud.Neotropical Fauna Environ. 32: 164–173.arcıa-Moreno, J., Arctander, P., and Fjeldså, J. (1998). Pre-Pliostecene differentiation among chat-tyrants. Condor 100: 629–640.arcıa-Moreno, J., and Fjeldså, J. (1999). A re-evaluation of specieslimits in the genus Atlapetes based on mtDNA sequence data. Ibis141: 199–207.ibbons, A. (1998). Calibrating the mitochondrial clock. Science 779:28–29.ilbert, D. G. (1992). SeqApp. A biosequence editor and analysisapplication, version 1.9a157. University of Indiana, Bloomington.ill, F. B. (1998). Hybridization in birds. Auk 115: 281–283.ackett, S. J. (1996). Molecular phylogenetics and biogeography oftanagers in the genus Ramphocelus (Aves). Mol. Phylogenet. Evol.5: 368–382.illis, D. M., Mable, B. K., and Moritz, C. (1996). Applications ofmolecular systematics: The state of the field and a look to thefuture. In ‘‘Molecular Systematics’’ (D. M. Hillis, C. Moritz, and B.K. Mable, Eds.), pp. 515–543. Sinauer, Sunderland, MA.ooghiemstra, H., Melice, J. L., Berger, A., and Shacketon, N. J.(1993). Frequency spectra and paleoclimatic variability of thehigh-resolution 30-1450 ka Funza I pollen reconr (Eastern Cordil-lera, Colombia). Quatern. Sci. Rev. 12: 141–156.

rwin, D. M., Kocher, T. D., and Wilson, A. C. (1991). Evolution of thecytochrome b gene of mammals. J. Mol. Evol. 32: 128–144.ishino, H., and Hasegawa, M. (1989). Evaluation of the maximumlikelihood estimate of the evolutionary tree topologies from DNAsequence data, and the branching order in Hominoidea. J. Mol.Evol. 29: 170–179.licka, J., and Zink, R. M. (1997). The importance of recent ice ages inspeciation: A failed paradigm. Science 277: 1666–1669.ocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Paabo, S.,Villafranca, F. X., and Wilson, A. C. (1989). Dynamics of mitochon-drial DNA evolution in animals: Amplification and sequencing withconserved primers. Proc. Natl. Acad. Sci. USA 86: 6196–6200.rajewski, C., and Fetzner, J. W. (1994). Phylogeny of cranes(Gruiformes: Gruidae) based on cytochrome-b DNA sequences. Auk111: 351–365.umar, S., Tamura, K., and Nei, M. (1993). MEGA: MolecularEvolutionary Genetic Analysis, version 1.01. University Park,

Pennsylvania, The Pennsylvania State University.

oewe, L., and Scherer, S. (1997). Mitochondrial Eve: The plotthickens. Trends Ecol. Evol. 12: 422–423.

osos, J. B., Warheit, K. I., and Schoener, T. W. (1997). Adaptivedifferentiation following eperimental island colonization in Anolislizards. Nature 387: 70–73.addison, W. P., and Maddison, D. R. (1992). MacClade: Analysis ofphylogeny and character evolution. Sunderland, Massachusetts,Sinauer Associates.aijer, S., and Fjeldså, J. (1997). A new Cranioleuca spinetail fromBolivia, with description of a ‘‘leapfrog pattern’’ of geographicalvariation in the genus. Ibis 139: 606–616.eyer de Schauensee, R. (1966). ‘‘The Birds of South America withTheir Distribution,’’ Livingston Press, Narbeth, PA.oore, W. S. (1995). Inferring phylogenies from mtDNA variation:Mitochondrial-gene trees versus nuclear-gene trees. Evolution 49:718–726.

acheco, J. F., Whitney, B. M., and Gonzaga, L. P. (1996). A new genusand species of furnariid (Aves: Furnariidae) from the cocoa-growingregion of southeastern Bahia, Brazil. Wilson Bull. 108: 397–606.

aterson, H. E. H. (1985). The recognition concept of species. In‘‘Species and Speciation’’ (E. S. Vrba, Ed.), pp. 21–29. TransvaalMuseum, Pretoria.

emsen, J. V. (1984). Geographic variation, zoogeography, and pos-sible rapid evolution in some Cranioleuca spinetails (Furnariidae)of the Andes. Wilson Bull. 96: 515–523.idgely, R. S., and Tudor, G. (1994). ‘‘The Birds of South America: TheSuboscine Passerines,’’ Oxford Univ. Press, London.oy, M. S. (1997). Recent diversification in African greenbuls (Pyc-nonotidae: Andropadus) supports a montane speciation model.Proc. R. Soc. Lond. B 278: 1–8.oy, M. S., Silva, J. M. C., Arctander, P., Garcıa-Moreno, J., andFjeldså, J. (1997). The speciation of South American and Africanbirds in montane regions. In ‘‘Avian Molecular Evolution andSystematics’’ (D. P. Mindell, Ed.), pp. 321–339. Academic Press,San Diego.

ilva, J. M. C. (1995). Biogeographic analysis of the South AmericanCerrado avifauna. Steenstrupia 21: 49–67.

tepien, C. A., and Kocher, T. D. (1997). Molecules and morphology instudies of fish evolution. In ‘‘Molecular Systematics of Fishes’’ (T. D.Kocher and C. A. Stepien, Eds.), pp. 1–11. Academic Press, SanDiego.

wofford, D. L. (1991). PAUP: Phylogenetic Analysis Using Parsi-mony, version 3.1.1. Illinois Natural History Survey, Champaign,Illinois.

an der Hammen, T., and Absy, M. L. (1994). Amazonia during thelast glacial. Palaeogeogr. Palaeoclim. Palaeoecol. 109: 247–261.

aurie, C. (1980). Taxonomy and geographical distribution of theFurnariidae (Aves, Passeriformes). Bull. Am. Mus. Nat. Hist. 166:1–357.

rba, E. S. (1992). Mammals as a key to evolutionary theory. J.Mamm. 73: 1–28.

ang, Z. (1996). Among-site rate variation and its impact on phyloge-netic analyses. Trends Ecol. Evol. 11: 367–372.

ink, R. M. (1996). Comparative phylogeography in North American

birds. Evolution 50: 308–317.