Molecular Phylogenetics and Evolution 90 (2015) 150–163
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Molecular Phylogenetics and Evolution
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Comparative phylogeography of co-distributed Phrygilus species (Aves, Thraupidae) from the Central Andes q ⇑ R. Álvarez-Varas a, D. González-Acuña b, J.A. Vianna a, a Departamento de Ecosistemas y Medio Ambiente, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Código Postal: 6904411, Casilla 306, Correo 22, Santiago, Chile b Facultad de Ciencias Veterinarias, Universidad de Concepción, Casilla 537, Chillán, Chile article info abstract
Article history: The Neotropical ecoregion has been an important place of avian diversification where dispersal and allo- Received 15 October 2014 patric events coupled with periods of active orogeny and climate change (Late Pliocene–Pleistocene) have Revised 10 April 2015 shaped the biogeography of the region. In the Neotropics, avian population structure has been sculpted Accepted 13 April 2015 not only by geographical barriers, but also by non-allopatric factors such as natural selection and local Available online 15 May 2015 adaptation. We analyzed the genetic variation of six co-distributed Phrygilus species from the Central Andes, based on mitochondrial and nuclear markers in conjunction with morphological differentiation. Keywords: We examined if Phrygilus species share patterns of population structure and historical demography, Central Andes and reviewed the intraspecific taxonomy in part of their geographic range. Our results showed different Comparative phylogeography Local adaptation phylogeographic patterns between species, even among those belonging to the same phylogenetic clade. Phrygilus P. alaudinus, P. atriceps, and P. unicolor showed genetic differentiation mediated by allopatric mechanisms ‘‘Sierra-finches’’ in response to specific geographic barriers; P. gayi showed sympatric lineages in northern Chile, while P. Allopatry plebejus and P. fruticeti showed a single genetic group. We found no relationship between geographic range size and genetic structure. Additionally, a signature of expansion was found in three species related to the expansion of paleolakes in the Altiplano region and the drying phase of the Atacama Desert. Morphological analysis showed congruence with molecular data and intraspecific taxonomy in most spe- cies. While we detected genetic and phenotypic patterns that could be related to natural selection and local adaptation, our results indicate that allopatric events acted as a major factor in the population dif- ferentiation of Phrygilus species. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction speciation scenarios have been proposed as potential explanations for avian diversification as a result of Andean orogenetic events The Neotropics supports the world’s richest avifauna popula- (Bonaccorso, 2009; Brumfield and Capparella, 1996; Burns and tions, constituting nearly a third of all living bird species (Rahbek Naoki, 2004; Campagna et al., 2011; Chaves et al., 2007; Weir and Graves, 2001). This remarkable species richness is thought to and Price, 2011). A number of studies have shown that be a consequence of processes associated with Andean orogenesis Pleistocene climate fluctuations may have induced alterations in and climatic fluctuations throughout the Late Pliocene– avian species distributions, population sizes and/or population Pleistocene, which also have contributed to sculpting the phylo- genetic structure (Bowie et al., 2006; Lougheed et al., 2013; geographic differentiation and population structure of diverse Lovette, 2005; Qu and Lei, 2009; van Els et al., 2012). Population Andean avian taxa (Bonaccorso, 2009; Campagna et al., 2011; genetic structuring is caused by restrictions of gene flow between Chaves et al., 2007; Fjeldså and Irestedt, 2009; Gutiérrez-Pinto populations and this can occur not only by geographical barriers or et al., 2012; Lougheed et al., 2013). Allopatric and parapatric isolation by distance, but also by non-vicariant factors such as nat- ural selection and adaptation to local environmental conditions (Chaves and Smith, 2011; Chaves et al., 2007; Cheviron and q This paper has been recommended for acceptance by C. Krajewski. Brumfield, 2009; Cheviron et al., 2005; Milá et al., 2009). In the ⇑ Corresponding author at: Departamento de Ecosistemas y Medio Ambiente, Central Andes, the opportunity for geographic isolation with diver- Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de gent selection associated with latitudinal and altitudinal habitat Chile, Av. Vicuna Mackenna 4860, Santiago, Chile. variation and climatic fluctuations is likely to generate intraspecific E-mail addresses: [email protected] (R. Álvarez-Varas), [email protected] (D. González-Acuña), [email protected] (J.A. Vianna). phenotypic and genetic divergence leading to differentiation and http://dx.doi.org/10.1016/j.ympev.2015.04.009 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163 151 ultimately speciation (Guarnizo et al., 2009; Kieswetter and In the present study we analyzed the genetic variation and mor- Schneider, 2013; Qu and Lei, 2009; Weir, 2006). phological differentiation of six co-distributed Phrygilus species Comparative phylogeography is a well-established field of bio- from the Central Andes, using mitochondrial and nuclear markers geography and study of speciation focused on describing evolu- in conjunction with morphologic data, to examine whether tionary divergence patterns found on a regional scale for Phrygilus species share patterns of population structure, lineage co-distributed populations of different taxa (Arbogast and divergences and historical demography. In addition, we reviewed Kenagy, 2001; Bermingham and Moritz, 1998). This is achieved the intraspecific taxonomy of these Andean species in most of their in two steps: first, by reconstructing the evolutionary histories of geographic range. two or more co-distributed species, and second, by determining whether parts of this shared history reflect common responses to 2. Materials and methods the same historical events (Victoriano et al., 2008). Theoretically, co-distributed avian species might have congruent phylogeo- 2.1. Sample collection and genetic data sets graphic patterns as a result of being subject to common environ- mental and geological changes. However, ecological adaptations A total of 106 blood samples from six species of the genus such as variations in altitudinal distribution, dispersal abilities or Phrygilus (P. alaudinus, P. atriceps, P. fruticeti, P. gayi, P. plebejus differences in food or habitat preferences would likely affect geo- and P. unicolor) were obtained from the brachial vein and preserved graphical patterns of morphological and genetic variation in ethanol. Birds were captured using mist nets following the proto- (Gavrilets, 2003; Morris-Pocock et al., 2010; Zhang et al., 2012). col of Gonzalez-Acuña et al. (2009). Samples were collected Phrygilus is a genus of Andean passerine birds that contains 11 between 2010 and 2013 from 9 localities from northern and central species found predominantly in open habitats across the Andes Chile along a latitudinal (18°Sto35°S) and altitudinal gradient and are distributed from Venezuela to Argentina and Chile (200–4800 m). We obtained a varied number of samples for each (Campagna et al., 2011; Jaramillo, 2005; Ridgely and Tudor, species (between 2 and 55 samples per species) and locality 1989). This genus of the family Thraupidae includes birds of graniv- (between 2 and 5 localities per species, see Supplementary Fig. 1). orous habits that have local migrations and possess variable latitu- Additionally, haplotypes from Peru and Argentina were obtained dinal and altitudinal ranges in their geographic distribution. Some from Genbank and included in our analyses (Campagna et al., species are found solely in the lowlands (e.g. P. carbonarius, P. patag- 2011; Kerr et al., 2009; Supplementary Fig. 1). The entire latitudinal onicus), some are altitude specialist (e.g. P. atriceps, P. dorsalis) and range of the study area comprised from Cajamarca in Peru (7°S) to others inhabit the complete altitudinal range (e.g. P. alaudinus, P Rio Negro in Argentina (41°S), covering lowland and highland envi- fruticeti, P. gayi). Moreover, some Phrygilus species have broad lati- ronments along the Andes (Fig. 1). For purpose of this paper we tudinal distribution along the Andes (e.g. P. plebejus, P. unicolor), grouped the localities into three major altitudinal regions: lowlands while others inhabit restricted geographic ranges (e.g. P. dorsalis, (sea level to 2400 m), pre-Puna (between 2400 and 3500 m) and P. erythronotus)(Campagna et al., 2011; Jaramillo, 2005). In addition Puna (over 3500 m) (Palma et al., 2005; Supplementary Fig. 1). to the geographical distribution variation, Phrygilus species also have marked morphological variation, which translates to a great variety of described subspecies in the literature, particularly those 2.2. DNA extraction, amplification and sequencing protocols that have a wide latitudinal distribution (Clements et al., 2013; Fjeldså and Krabbe, 1990; Goodall et al., 1946). These features sug- DNA was isolated from blood samples using a salt protocol gest these Andean species can likely serve as good candidates for developed by Aljanabi and Martinez (1997). We amplified two comparative phylogeographic studies, allowing examination of mitochondrial regions, the cytochrome oxidase I (COI) and the con- how vicariant and non-vicariant events may have influenced their trol region (CR). In addition we amplified the fifth intron of the diversification and distribution in the Central Andes. nuclear beta-fibrinogen gene (Fib5). Primers were those as Phylogenetic reconstruction of the genus Phrygilus using mito- described in Kerr et al. (2009) and Kimball et al. (2009). The frag- chondrial and nuclear markers suggests an origin of the species ment size for each marker varied among species (Table 1). The mainly in the Pleistocene, with representatives diversifying within, reactions were carried out in 25 ll volume containing 2 ll DNA, out of, and into the Andes (Campagna et al., 2011). These same 1Â reaction buffer, 200 lM of each dNTP, 0.5 lM of each primer authors concluded that the genus is polyphyletic, comprising four and 1 unit Taq DNA polymerase Platinum (Invitrogen). The MgCl2 distantly related main clades with at least nine other genera inter- concentrations varied among species from 1.5 to 2.0 mM and we spersed. These phylogenetic clades agreed with the grouping of followed the thermal cycle profile described in Campagna et al. Phrygilus species based on plumage traits proposed by Ridgely (2011). PCR products were visualized using electrophoresis on 1% and Tudor (1989), suggesting single evolutionary origins for each. agarose gels with GelRed Nucleic Acid Stain (Biotium), purified Moreover, Campagna et al. (2011) observed that especially those and sequenced bi-directionally at Macrogen Inc., Seoul, South species with broadest altitudinal and latitudinal distributions Korea. All sequences were deposited in Genbank accession num- showed deep genetic splits and phylogeographic structure, deserv- bers: KM891674-KM891724 for Fib5, KP015134-KP015174 for ing further research. COI and KR076824-KR076864 for CR. We evidenced several According to the above, we expect that Phrygilus species with heterozygote sites for CR sequences, indicating heteroplasmy broad geographic distribution range and marked morphological caused by mtDNA duplication or nuclear sequences of mitochon- variation should exhibit pronounced genetic structure in accor- drial origin (numts) as observed in other avian species (e.g. dance with landscape heterogeneity and presence of geographical Abbott et al., 2005; Kumazawa et al., 1996). CR sequences were barriers, in contrast to geographically restricted and monotypic only obtained for Phrygilus fruticeti (n = 17) and P. alaudinus species. We also expect those species belonging to the same phylo- (n = 24), therefore were not included in the analysis. genetic clade to show similar demographic history and a common phylogeographic pattern. In the same way, we expect that Andes 2.3. Phylogenetic analyses orogeny, other physical barriers (desert, water sources, and deep valleys) and climatic fluctuations that occurred during Sequences were aligned and polymorphic sites were confirmed Pleistocene to sculpt the intraspecific genetic diversity of by eye according to the chromatogram using Sequencher 5.1. Phrygilus species in the Central Andes. (Gene Codes Corporation, Ann Arbor, MI, USA). Polymorphic sites 152 R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163
Fig. 1. Distribution of Phrygilus species (shaded gray areas) and sampled geographic locations (black dots). (a) P. alaudinus, (b) P. atriceps, (c) P. fruticeti; (d) P. gayi, (e) P. unicolor and (f) P. plebejus. Distribution based on IUCN data.
Table 1 To detect intraspecific lineages, phylogeny was reconstructed Molecular indices from mtDNA cytochrome oxidase I (COI) for Phrygilus species and for COI (437 bp), Fib5 (551 bp) and the concatenated marker intraspecific clusters. (COI + Fib5, 956 bp) using a total of 164 sequences, with Diuca Species Cluster NS h Hd p DFs diuca as outgroup (Accession numbers FJ027510.1 and P. alaudinus – 62 27 18 0.88 0.006 À1.20 À4.68* JN417975.1 for COI and Fib5, respectively). We constructed phylo- 630 bp A 55 14 15 0.85 0.002 À1.50* À9.07** genetic trees through Bayesian methods using Mr Bayes 3.1.2 B 6 1 2 0.53 0.0008 0.85 0.63 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, C101––– – 2003). The evolutionary model for each marker was selected using P. atriceps – 17 8 6 0.83 0.003 À0.94 À0.96 jModelTest 0.1.1 (Guindon and Gascuel, 2003; Posada, 2008) and 584 bp A 16 4 5 0.82 0.002 0.27 À0.75 Akaike Information Criterion (AIC). The best-fit models of nucleo- B101––– – tide substitution were HKY + G for COI, GTR + G for Fib5 and ** ** P. fruticeti – 46 14 15 0.70 0.002 À2.14 À14.55 HKY + I for the concatenated tree. The Bayesian analyses were 520 bp A 26 5 6 0.64 0.002 À1.02 À2.39 B 20 10 10 0.76 0.002 À2.15** À8.08** run for one million generations, sampling every 100 generations. At this point the standard deviation of split frequencies was P. gayi – 14 17 8 0.89 0.007 À0.94 À0.98 559 bp A 2 2 2 1 0.004 0 0.69 <0.01, indicating that both runs had converged. Additionally the B 12 6 6 0.85 0.003 À0.70 À1.90 Potential Scale Reduction Factor (PSRF) was very close to one for P. plebejus – 16 8 7 0.75 0.003 À1.70* À2.44* all parameters, indicating that we had adequately sampled their 522 bp A 10 8 7 0.91 0.004 À1.57* À3.13** posterior distributions. Finally, to create a consensus tree we used B 6 1 2 0.33 0.0006 À0.93 À0.003 Figtree 1.2.2 (Rambaut, 2009). P. unicolor – 9 28 8 0.97 0.023 1.04 À0.61 526 bp A 3 6 3 1 0.008 0 0.13 2.4. Estimation of diversification times B 6 5 5 0.93 0.004 À0.47 À1.97
N: sample size; S: number of polymorphic sites; h: haplotype number; Hd: gene We estimated interspecific and intraspecific node ages using a diversity; p: nucleotide diversity; D: Tajima’s D values; Fs: Fu’s Fs values. Bayesian approach implemented in the BEAST 1.4.8 (Drummond * <0.05. and Rambaut, 2007) and Figtree 1.2.2 (Rambaut, 2009). To obtain ** <0.01. absolute times we used the cytochrome oxidase I (COI) data set and haplotypes were identified using ClustalX 2.1 (Thompson et al., and a calibration of 2.1% per million years (Weir and Schluter, 1997). To identify haplotypes of heterozygotes in the nuclear 2008). The analysis was run for 100 million generations using intron sequences we used Phase, a Bayesian approach imple- HKY + G model of nucleotide substitution with four rate categories, mented in the DNAsp 5.10.1 program (Rozas, 2009). assuming a constant population size and a relaxed uncorrelated R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163 153 lognormal clock. Using Tracer 1.0.1 (Rambaut and Drummond, all intraspecific clusters the best model found by jModelTest was 2007), we checked for convergence in parameter estimations by HKY. Analyses were run for 20 millon iterations, sampling every verifying that trends were not observed in traces and that effective 1000 steps and assuming a substitution rate of 2.1% per million sample sizes were adequate. years (Campagna et al., 2011).
2.5. Genetic units analysis 2.7. Morphological analysis
We evaluated intraspecific genetic structure with COI and To investigate the relationship between genetic and morpho- COI + Fib5 sequence data using the program Geneland 1.0.7 in logical variation and test whether there is congruence between the R-package (Guillot et al., 2005), which implements population these tools to discriminate subspecies, we made morphological statistical models with Bayesian inference in a set of measurements on 239 individuals. This information was obtained geo-referenced individuals with sequence data. This model infers only for birds from Chilean localities (the same localities used for and locates the genetic discontinuities between populations of genetic analyses; Supplementary Fig. 1). We did not include sam- geo-referenced genotypes, considering the uncertain localization ples from Peru and Argentina. Six measurements were taken using of the sampled individuals (Palma et al., 2012). Finally, this analy- dial calipers to an accuracy of 0.1 mm: total length, bill depth, wing sis allows inferring specific genetic clusters with grouped length, tarsus length, tarsus width and tail length. In addition, body sequences. The parameters for this analysis were: a maximum rate mass was recorded using a Voltcraft HS-30 Digital Scale. This last of the Poisson process fixed at 100, uncertainty of the spatial loca- measurement was discarded from our analysis because it varied tion fixed at 5 km and the maximum number of nuclei in the at different times of day. The variance in each variable deviated Poisson-Voronoi tessellation fixed at 300; we used 5 million from a normal distribution (Shapiro–Wilk test) and therefore we Markov Chain Monte Carlo (MCMC) iterations. These parameters used log-transformed data in all subsequent statistical analyses. were used for five repetitions of K-values (number of clusters) in To remove the effect of overall size and to analyze shape, we stan- the range 1–9. Using the same parameters and the K-values dardized each character against total length and used the residuals inferred above as a fixed variable, the MCMC algorithm was run as variables. Principal component analysis (PCA) was applied to all 30 times. The mean logarithm of the posterior probability was cal- six log-transformed morphological variables as well as to the five culated for each of the 30 runs, and the posterior probability of size-adjusted residuals. The corrected and transformed measure- population membership for each pixel of the spatial domain was ments were all correlated, so all were used in a principal compo- computed for the three runs with the highest values. nents analysis. A one-way analysis of variance (ANOVA) among With information recovered from the Geneland program we populations for each species was performed on factor scores from grouped populations into clusters and estimated the genetic diver- the first two principal-components. PCA analyses were performed sity of COI sequences calculating the following summary statistics in Past: Paleontological Statistics 2.17 (Hammer et al., 2001) and in Arlequin 3.5.1.2 (Excoffier and Lischer, 2010): number of poly- ANOVA was performed using the software R (R Development morphic sites (S), haplotype number (h), gene diversity (Hd) and Core Team, 2013). nucleotide diversity (p), as well as Tajima’s D and Fu’s Fs to assess deviations from neutrality. These genetic parameters were also cal- culated for each species. We also assessed the amount of genetic 3. Results structure by performing analyses of molecular variance (AMOVA) 3.1. General genetic patterns of variation and calculating pairwise genetic differences (FST) in Arlequin 3.5.1.2 (Excoffier and Lischer, 2010) for each pair of localities and A total of 106 individuals were sequenced for COI, representing intraspecific clusters. Molecular indices, AMOVA and FST were not calculated from COI + Fib5 sequences since the sample size and 9 localities from northern and central Chile. Additionally 10 sam- number of localities decreased (in comparison with COI). ples from Peru and 48 samples from Argentina were obtained from Furthermore, Geneland analysis did not show clear patterns and Genbank comprising a total of 164 samples (see Supplementary clusters definition for all species. Fig. 1). In the case of Fib5, we sequenced 91 individuals from the Finally, in order to visualize genetic diversity and possible geo- same Chilean localities and obtained 10 samples from Peru and graphic associations among haplotypes, we constructed median 11 from Argentina from Genbank (see Supplementary Fig. 1). The joining networks (MJN) from COI and COI + Fib5 sequences in concatenated mtDNA and nuclear marker comprised a total of 93 NETWORK 4.6.1.1 (Bandelt et al., 1999). samples. Molecular indices were calculated for COI for all species and intraspecific clusters (Table 1). We recovered 18 unique haplo- 2.6. Demographic history types for P. alaudinus, 15 for P. fruticeti, 8 for P. gayi and P. unicolor, 7 for P. plebejus and six haplotypes for P. atriceps. The number of We studied Phrygilus historical demography by calculating the polymorphic sites (S) in the different species varied between 8 mismatch distribution for every species and cluster with a signa- and 27, being highest in P. alaudinus and lowest in P. atriceps and ture expansion (significant values of Tajima’s D and/or Fu’s Fs). P. plebejus. The haplotype diversity (Hd) was moderate to high, These analyses were performed in Arlequin 3.5.1.2 (Excoffier and varying between 0.70 and 0.97. Nucleotide diversity (p) varied Lischer, 2010) with 1000 bootstrap replicates to assess signifi- between 0.002 and 0.023. P. unicolor showed the highest values cance. In addition, we looked for evidence of historical signatures and P. fruticeti the lowest values for both haplotype and nucleotide of fluctuations of population sizes by examining the Bayesian diversity. Skyline Plot and Bayesian Skyride Plot (BSP) in BEAST 1.4.8 (Drummond and Rambaut, 2007) and Tracer 1.0.1 (Rambaut and 3.2. Intraspecific phylogeographic structure Drummond, 2007). The BEAST program depicts a coalescent-based estimation of population size changes over time For P. alaudinus, Geneland analysis from COI sequences showed with an MCMC sampling procedure. Analyses were performed for three genetic clusters (k = 3; A, B and C), which agreed with the every species and intraspecific cluster with marked divergence fragmented distribution of this species including populations from (based on MJN results). We estimated the substitution model using Peru (pre-Puna), Chile (lowlands) and Argentina (pre-Puna) sepa- jModelTest 0.1.1 (Guindon and Gascuel, 2003; Posada, 2008). For rately (Fig. 2). P. atriceps and P. unicolor showed similar patterns 154 R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163
Fig. 2. Geneland analysis based on mtDNA data (COI) showing the posterior membership probability of individuals of the genus Phrygilus in Chile, Peru and Argentina. (a) P. alaudinus, (b) P. atriceps, (c) P. fruticeti; (d) P. gayi, (e) P. unicolor and (f) P. plebejus. Blue dots represent Cluster A, red dots indicate Cluster B and green dots Cluster C (the dark red dot in b) represents a site with 0.5 membership probability). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). with two genetic clusters (k = 2; A and B), separating Peruvian pop- ulations (pre-Puna and Puna) from populations of the Chilean Table 2 Pairwise FST values (below diagonal) and their respective p values with standard Altiplano (Puna) and northwestern Argentina (pre-Puna and deviation (above diagonal) from intraspecific clusters using data from mtDNA Puna). In the case of P. fruticeti, Geneland analysis showed two cytochrome oxidase I (COI). clusters (k = 2; A and B) separated by latitude and the central local- Species Cluster A B C ity that had a low group membership probability (Fig. 2). Finally in P. gayi and P. plebejus the Llanos del Challe (lowlands) and Chilean P. alaudinus A – 0.00 ± 0.00 0.99 ± 0.0002 B 0.88** – 0.99 ± 0.0002 Altiplano (Puna) populations remained isolated from the rest of the C 0.88 0.94 – populations, generating two intraspecific clusters (k = 2; A and B; P. atriceps A – 0.02 ± 0.001 – Fig. 2). Analysis performed with concatenated sequences B 0.74* –– (COI + Fib5) showed two genetic clusters in all species except for P. fruticeti A – 0.07 ± 0.003 – P. gayi (k = 3; see Supplementary Fig. 2). P. alaudinus showed clus- B 0.05 – – ters separating Chile from Peru and Argentina populations (these P. gayi A – 0.03 ± 0.001 – two last belonging to the same cluster); P. unicolor showed a sim- B 0.85* –– ilar pattern as COI, with two clusters separating Peruvian popula- P. plebejus A – 0.06 ± 0.008 – tions of the Chilean Altiplano and northwestern Argentina. B 0.15 – – Finally, P. atriceps, P. fruticeti, P. gayi and P. plebejus showed clusters P. unicolor A – 0.01 ± 0.002 – with low group membership probabilities and without a clear pat- B 0.88** –– tern of differentiation. * <0.05. Pairwise genetic differences (FST) were calculated among ** <0.01. intraspecific clusters and AMOVA by considering localities and clusters from COI data. Our results showed high and significant val- ues of FST in the case of P. alaudinus, P. atriceps, P. gayi, and P. uni- The Bayesian phylogeny for mitochondrial (COI) and concate- color between Cluster A and B (Table 2). Additionally, these species nated gene trees (COI + Fib5) revealed similar pattern to that found showed more variation among groups (clusters) and FST values in the median joining networks (MJN) for the same markers, with from AMOVA were significant in all cases (data not shown). In con- polytomy for P. alaudinus, P. fruticeti and P. plebejus; and two clades trast, P. fruticeti and P. plebejus showed non-significant FST values for P. atriceps, P. gayi, and P. unicolor (Figs. 3 and 4 and between clusters and greater variation within populations (locali- Supplementary Fig. 2 respectively). In all species we obtained an ties regardless of clusters) without significant FST in AMOVA. MJN coincident with Geneland clusters based on COI sequences, R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163 155 except for P. fruticeti and P. plebejus, where the MJN showed a sin- supported the occurrence of two subspecies, but the morphological gle genetic group. In the cases of P. alaudinus, P. atriceps, P. gayi, and data just supported one subspecies. P. unicolor we detected divergent haplogroups well separated by several mutation steps (Fig. 4). In contrast, P. fruticeti and P. plebe- 4. Discussion jus showed a star-like network topology represented by one highly frequent central haplotype and various private haplotypes (Fig. 4). 4.1. Complex genetic diversity patterns in Neotropical birds For all species, MJN from COI + Fib5 was more concordant with MJN from COI than with Geneland results from concatenated data Our results from mitochondrial and nuclear sequences showed since this last analysis did not show clear patterns in most species different patterns of genetic variation between the Phrygilus spe- (Fig. 4 and Supplementary Fig. 2). cies studied, even among those belonging to the same clade or plu- mage group (Campagna et al., 2011; Ridgely and Tudor 1989). P. 3.3. Diversification times unicolor (Clade I), P. alaudinus (Clade II), P. gayi and P. atriceps (Clade III) showed higher haplotype and nucleotide diversity and Species and internal node dates estimated by BEAST are indi- deep divergence in all tree topologies. P. gayi showed sympatric cated in Fig. 3, including upper and lower limits of 95% confidence lineages in northern Chile, while the other three species showed intervals. Our results showed more recent divergence times genetic differentiation mediated by allopatric mechanisms in between species than obtained by Campagna et al. (2011). This response to specific geographic barriers. In contrast, P. plebejus was probably due to the fact that Campagna et al. (2011) used all and P. fruticeti (Clade I and II respectively) had lower genetic diver- species of the genus in their study, and included even other avian sity, slight intraspecific divergence and a single genetic group genera for this estimation. Diversification time for Clade II was without differentiation. found to be approximately 1.8 Mya, followed by clade I at 1.1 Diverse patterns of genetic variation using COI sequences in Mya and finally Clade III at 0.67 Mya. At the intraspecific level, Neotropical birds were reported by Kerr et al. (2009), when com- Chilean and Argentinian populations of P. alaudinus diverged pared to those of North American birds. Most of the genetically approximately 0.3 Mya, while populations from Argentina and divergent groups in North America reflected east–west allopatry Peru diverged at 0.2 Mya (Cluster A, B and C; Fig. 3). Peruvian pop- (Kerr et al., 2007), while divergences in Argentina were more com- ulations of P. atriceps diverged from Chilean Altiplano (Puna) and plex; some are north–south, others are east–west and yet others northwestern Argentina populations approximately 0.2 Mya. In occur along altitudinal gradients or in response to specific habitat the case of P. gayi, populations from Llanos de Challe (Cluster A) barriers, involving parapatric or sympatric lineages. These results diverged from the remaining populations approximately 0.15 indicate that although the ages of species appear similar in temper- Mya. Finally, for P. unicolor, Peruvian (Cluster A) and Chilean and ate North and South American avifauna, patterns of regional diver- Argentinian (Cluster B) populations diverged from one another gence are more complex in the Neotropics, suggesting that the high approximately 0.72 Mya. diversity of Neotropical avifauna has been fueled by greater oppor- tunities for regional divergence. The Neotropics has been recog- 3.4. Demographic history nized as an important place of avian diversification in which both dispersal and vicariance mechanisms coupled with periods Neutrality tests were calculated to detect evidence of popula- of active orogeny and climate changes in the Late Pliocene– tion expansion for every intraspecific cluster. In the cases of P. Pleistocene shaped the biogeographic scenario of the ecoregion alaudinus (Cluster A), P. fruticeti (single group) and P. plebejus (sin- (Chaves et al., 2007; Lougheed et al., 2013). In South America, these gle group) we found a signature of population size expansion (neg- factors may have allowed the formation of dry, sparsely vegetated ative values of Fu’s Fs and Tajima’s D). In contrast, P. atriceps, P. areas such as the Andean Altiplano, the Atacama Desert and unicolor and P. gayi did not show expansion signatures (Table 1). Patagonia, thus providing new environments for the differentiation P. alaudinus showed a bimodal mismatch distribution, P. fruticeti of local biota (Palma et al., 2005). a unimodal distribution and P. plebejus slightly bimodal distribu- The varied genetic patterns found in Phrygilus species in this tion (Fig. 5). The results obtained from BEAST (BSP, Bayesian study are concordant with the geological and environmental his- Skyline or Skyride Plot) showed an increment in the effective pop- tory of the Neotropical region associated with vicariant events ulation size starting about 15,000 years ago in lowlands for P. and climate changes. Campagna et al. (2011), who reconstructed alaudinus and P. plebejus and P. fruticeti about 60,000 years ago in a complete phylogeny of the genus Phrygilus, found that its diver- highlands (Fig. 5). sification in South America was mainly associated with Pleistocene glaciations or concomitant vegetation shifts. Similarly, Lougheed 3.5. Variation in morphological traits et al. (2013) studying another Neotropical passerine bird broadly distributed in the American continent, the Rufous-collared sparrow For these analyses we included only samples from Chilean (Zonotrichia capensis) suggested that its evolutionary history was localities to evaluate if morphological and genetic data are congru- sculpted by changes associated with Pleistocene biogeographic ent in terms of subspecies delimitation. We did not include sam- events. ples of P. unicolor due to small sample size. Eigen values and percentage of variation explained by the first two principal-components from log-transformed and corrected data 4.2. Genetic structure and geographic range for each species are shown in Table 3. ANOVA of the factor scores from the first and second PC axes, with log-transformed and cor- Recent studies performed in Neotropical birds have demon- rected data, did not show significant differences among population strated that species with broad geographic ranges have highly in Phrygilus species (Table 3). These results suggest that there is no structured populations (Cadena et al., 2007; Chaves and Smith, differentiation in morphological traits among Chilean localities for 2011; Cheviron et al., 2005; Lougheed et al., 2013). Furthermore, Phrygilus species. Furthermore, our results showed coincidence Campagna et al. (2011) using mitochondrial and nuclear sequences between morphological and genetic information in all cases for in Phrygilus, observed that species with broad altitudinal and lati- log-transformed and corrected data (one subspecies detected, tudinal distributions showed phylogeographic structure. These Fig. 6). The only exception was P. gayi, in which the genetic results findings generally have been associated with ecological factors 156 R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163
Fig. 3. Bayesian phylogenetic reconstruction of the genus Phrygilus. Posterior probability support values are shown at nodes. (a) mtDNA data (COI, 437 bp) with node dates and confidence interval and (b) concatenated data (COI-Fib5, 956 bp). such as dispersal capacity and/or presence of barriers to gene flow (2011) studied a widespread aquatic songbird (Chrysomus ictero- and environmental heterogeneity (Cadena et al., 2011; Chaves and cephalus) finding no detectable genetic structure across its range, Smith, 2011; Fernandes et al., 2013; Gutiérrez-Pinto et al., 2012; including lowlands and highlands habitats of South America. Lougheed et al., 2013). Our results showed no relationship between These results were attributed especially to ecological features geographic range size and genetic structure in Phrygilus species. In related to the high dispersal capacity of this species and hence high this study, P. unicolor, P. alaudinus and P. gayi, which have broad gene flow between populations. In our case, although P. fruticeti geographic distributions (occurring in different types of ecosys- and P. plebejus possess a broad geographic distribution and shared tems), showed a pattern of genetic variation similar to P. atriceps, barriers that limit gene flow in other Phrygilus species (e.g. Titicaca the species with the most restricted geographic distribution (only Lake, mentioned below) we found no phylogeographic break. highlands). This pattern was characterized by well-defined genetic Given the above, this genetic pattern is possibly due to biological clusters (with high membership probabilities in Geneland), signif- or ecological characteristics associated with dispersal abilities or icant FST values and markedly divergent haplogroups in the MJN. more generalist habitat/food preferences in both species, which Conversely, P. plebejus and P. fruticeti, species with wide distribu- differs from the rest of Phrygilus species. The genus Phrygilus is tion both latitudinally and altitudinally, and whose sampling characterized by having a granivorous diet (Estades and Estades, incorporated much of their ranges, did not exhibit divergence 1997; Estades and Temple, 1999; González-Gómez et al., 2006; between haplotypes in the MJN for the genetic groups defined in Mcgehee and Clinton, 2007; Vuilleumier, 1994) and occurring in
Geneland. Additionally, we found non-significant FST values in open habitats (Fjeldså and Krabbe, 1990; Jaramillo, 2005; Ridgely these species, suggesting undifferentiated populations and high and Tudor, 1989); however, there is still a lack of information gene flow among studied locations. Although a number of studies about the characteristics of each species, including dispersal capa- have described marked genetic structure in populations of passer- bilities, feeding adaptations, etc. If we consider that diet and habi- ines widely distributed in America (Chaves and Smith, 2011; tats are shared between the six Phrygilus species studied, it is García-Moreno et al., 2004; Weir et al., 2008), Cadena et al. probable that dispersal capacities are different in P. plebejus and R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163 157
Fig. 4. Median joining network for Phrygilus species from mtDNA cytochrome oxidase I data (COI; between 520 bp and 630 bp). (a) P. alaudinus, (b) P. atriceps, (c) P. fruticeti; (d) P. gayi, (e) P. unicolor and (f) P. plebejus. More than six mutation sites are indicated with a square with the corresponding number of mutated positions.
P. fruticeti with respect to other species, thus leading to more populations were isolated from other sampled locations in the genetically homogenous populations. Although Dalmas and highlands of Chile and Argentina. In addition, we found two genetic Negrin (2011) mentioned more extensive migrations in P. fruticeti, clusters which were highly divergent and with higher number of to date there are few data about the movements of all Phrygilus mutational steps for P. unicolor. The estimated divergence time species, highlighting the need for increasing research on basic between populations of this species was of 0.7 Mya, and 0.2 Mya aspects related to the biology and ecology of these Andean birds. between populations of P. atriceps. This marked genetic differenti- ation in the Peruvian Altiplano may be associated with Pleistocene 4.3. Comparative phylogeography glacial cycles, where contraction and expansion occurred in what are known as paleolakes (Kroll et al., 2012; Placzek et al., 2009), Most Phrygilus species showed a marked phylogeographic pat- especially considering the estimated divergence date for both spe- tern, however, breaks were varied between species and those with cies. A variety of studies have described the role of courses of water similar evolutionary history (e.g. belonging to the same clade) did (rivers, lakes, etc.) as barriers to the dispersal of Neotropical birds not necessarily show the same pattern. Estimated divergence times (Bonaccorso, 2009; Chaves and Smith, 2011; Fernandes et al., 2013; also were variable among clades and species. P. unicolor show dee- Gutiérrez-Pinto et al., 2012). Lake Titicaca is a freshwater lake sit- per divergence times in relation to the remainder of species uated in the northern portion of the Altiplano (Bolivia, Peru) at an (0.72 Myr ago). Moreover, this intraspecific divergence age is elevation of 3810 m above sea level. Its modern area encompasses 2 greater than the interspecific divergence age in Clade III, which is approximately 8500 km (Fritz et al., 2007; Dejoux and Iltis, 1992); composed of P. gayi and P. atriceps (0.67 Mya). P. unicolor and P. it is more than 284 m deep (Argollo and Mourguiart, 2000). The atriceps, belonging to different phylogenetic clades (Clade I and lake originated during the Late Pliocene/Early Pleistocene, about III, respectively), shared a common phylogeographic pattern and 2–3 million years ago (Lavenu, 1992) and underwent at least five a break in similar locations. In both species, Peruvian Altiplano major phases of expansion and contraction during the Late 158 R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163
Fig. 5. Mismatch distribution and BSP analysis from mtDNA cytochrome oxidase I (COI) data for (a) P. alaudinus, (b) P. fruticeti and (c) P. plebejus.
Pleistocene, increasing its water levels in glacial periods and flow for both species. Vicariance has been proposed as the main decreasing during interglacial periods (Argollo and Mourguiart, process which generated the diversification of the flora and fauna 2000; Fritz et al., 2007; Kroll et al., 2012). The high genetic differ- of the Altiplano region, probably stimulated by processes such as entiation found in populations of P. atriceps and P. unicolor north the Andes uplift, intense volcanic activity, multiple cycles of pale- and south of Lake Titicaca using a mitochondrial marker (COI) sug- olakes formation and the Pleistocene glaciations (Kroll et al., 2012; gests that this lake may have acted as a historical barrier to gene Sáez et al., 2014; Vila et al., 2013). Recent studies in other taxa R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163 159
Table 3 Eigen values, percentage of variance and one-way analysis of variance (ANOVA) of the factor scores from the first and second PC axes, with corrected and transformed data of Phrygilus species.
Species Corrected data Log-transformed data PC1 PC2 PC1 PC2 Eigen value %variance ANOVA Eigen value %variance ANOVA Eigen value %variance ANOVA Eigen value %variance ANOVA P. alaudinus 1.97 39.46 2.657 1.09 21.91 0.041 1.7 28.35 0.853 1.22 20.48 0.649 P. atriceps 2.26 45.17 0.075 1.61 32.27 0.102 2.34 39 0.027 1.65 27.49 1.166 P. fruticeti 2.41 48.12 0.023 1.07 21.37 4.871 2.52 42.01 0.128 1.24 20.62 2.23 P. gayi 1.46 29.21 1.33 1.43 28.54 0.77 2.12 35.29 4.077 1.36 22.7 0.187 P. plebejus 2.18 43.65 4.639 1.12 22.36 0.396 1.51 25.09 1.316 1.32 22 0.187
Fig. 6. Principal component analysis of morphological data for Phrygilus species. (a) Five corrected morphological variables (size-adjusted) and (b) six log transformed morphological variables in 239 specimens from Chilean localities. inhabiting this region suggested genetic differentiation mediated differentiation found between populations north and south of by allopatric (e.g. fishes, Vila et al., 2013, snails, Collado et al., Lake Titicaca suggest an allopatric pattern of diversification in 2011) and parapatric mechanisms (e.g. rodents, Palma et al., the Altiplano region, derived mainly from glacial and interglacial 2005; amphibians, Sáez et al., 2014). In our case, the genetic cycles associated with the Pleistocene. Sarno et al. (2004) 160 R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163 performed a phylogeographic study in Vicuña populations clusters. Nevertheless, this can be attributed to a pattern of isola- (Camelidae) in the Chilean and Bolivian Altiplano suggesting that tion by distance rather than a phylogeographic boundary. This is the Andes Range and paleolake formation could have restricted supported by a central locality with low membership probability gene flow between Chilean and northern Bolivian populations in in Geneland and the star-like MJN topology, showing closely the past. Although we do not have more evidence about Lake related haplotypes shared among localities and private haplotypes Titicaca as a barrier for other terrestrial vertebrate taxa, our results that were shared between nearby locations. However, the small support this lake as an important barrier to gene flow at least for sample sizes obtained from each locality did not allow perfor- populations of P. unicolor and P. atriceps, highlighting the impor- mance of Mantel test to evaluate the pattern of isolation by dis- tance of increasing comparative phylogeographic studies in this tance in this species. region. In contrast to the rest of species, in Phrygilus alaudinus we found P. gayi showed an interesting pattern; we observed a genealog- three highly structured intraspecific clusters with marked diver- ical break in the absence of a contemporary physical barrier to gence among them, where the separation was clearly defined by gene flow. A large number of studies have demonstrated the the discontinuous range of the species and the Andes Range importance of physical barriers in avian population structure (Cluster A: lowlands in Chile; Cluster B: pre-Puna of Argentina (Arbeláez-Cortés et al., 2010; Barrera-Guzmán et al., 2012; and Cluster C: pre-Puna of Peru). Populations from Chile and Bonaccorso, 2009; Chaves and Smith, 2011; García-Moreno et al., Argentina diverged approximately 0.3 million years ago and those 2004; Nicholls and Austin, 2005). However, increasing evidence from Argentina and Peru approximately 0.2 million years ago. P. in varied taxa has suggested that phylogeographic breaks can form alaudinus inhabits from sea level up to 3500 m (Fjeldså and without contemporary or historical geographical barriers (e.g. Krabbe, 1990; Ridgely and Tudor, 1989), although subspecies P. a. birds, Arbeláez-Cortés et al., 2014; Irwin, 2002; frogs, Guarnizo excelsus (Peru and Bolivia) reaches up to 4100 m (Fjeldså and et al., 2009). Additionally, restrictions to gene flow may be caused Krabbe, 1990). The fragmented distribution of its populations in not only by geographic barriers, but also by non-vicariant factors Peru, Chile and Argentina agrees with its altitudinal restriction such as natural selection acting on fitness traits when environmen- mediated by the mountain range, which exceeds 6000 m in this tal conditions and selective regimes differ sufficiently between region (Zeil, 1979). This distribution of genetic diversity is similar localities (Milá et al., 2009). Our analyses showed an isolation of to that found in other Andean biota (e.g. fishes, Ruzzante et al., the Llanos del Challe population in the Atacama Desert (lowlands) 2006; butterflies, Brower, 1996; frogs, Bernal et al., 2005; lizards, in relation to the rest of the populations (including the Fray Jorge Victoriano et al., 2008) with highly structured variation along both population, which is located about 300 km south of Llanos de the eastern and western slopes of the Central Andes. This allopatric Challe, a semi-desertic area). Divergence between these popula- pattern has also been seen in other Neotropical birds such as tions was estimated to be approximately 0.15 Mya. Nevertheless Andean hummingbirds of the genus Adelomyia in southern Peru we did not detect current or historical topographical barriers and Ecuador, where genetically divergent populations were found (e.g. rivers, mountains) or other studies that support local specific on the two sides of the Andes whose separation occurred during conditions in this area that could genetically separate these popu- the final stages of the Andean uplift in the Pliocene (Chaves and lations. To date, three subspecies have been described for P. gayi in Smith, 2011; Chaves et al., 2007). Divergent populations were also Chilean literature: P. g. gayi, inhabiting mainly highlands between found in Glyphorynchus spirurus, a tropical forest bird; the Andes Atacama (26°S) and Chillán (36°S); P. g. minor, distributed in the acted as a barrier to gene flow between ancient subspecific lin- coastal zone between Chañaral (26°S) and Santo Domingo (33°S) eages (Milá et al., 2009). In the same way, Parra et al. (2009) found and P. g. caniceps, between Aysén (45°S) and Tierra del Fuego that closely related species of birds belonging to the Coeligena (55°S) (Clements et al., 2013; Fjeldså and Krabbe, 1990; Goodall genus underwent either allopatric or parapatric speciation on et al., 1946). Although P. g. gayi occurs in mountain and foothill opposite Andean slopes, indicating the importance of geographic areas in northern and central Chile, during winter periods individ- isolation for speciation in this group. Our results suggest that the uals migrate to the lowlands, even reaching the coast (Goodall Andes generate isolation on populations of P. alaudinus leading to et al., 1946) where they would be sympatric with P. g. minor.Itis a marked intraspecific divergence, especially among Chilean and probable that the pattern of genetic differentiation observed in Argentinian populations (Cluster A and B). This divergence had Llanos de Challe is due to samples obtained from different sub- an estimate date of approximately 0.3 million years ago, when species, which diverged 0.15 Myr ago. Given that the sampling in the Andes had already reached its current elevation (Armijo Fray Jorge was performed during winter season (when P. g. gayi et al., 2015). Therefore, our findings allow us to suggest that P. migrates to the coast) and that Cluster B was composed of haplo- alaudinus could be going through a peripatric speciation process, types from the northern coast (Fray Jorge) and southern Chilean at least among populations from Chile and Argentina. In this case, countryside (Termas del Flaco), these individuals might correspond the Andes range would limit gene flow, thus playing a fundamental to the subspecies P. g. gayi. Conversely, individuals sampled in role in this intraspecific divergence. Nevertheless, we did not Llanos de Challe would probably correspond to P. g. minor, which observe the same pattern in the remaining Phrygilus species in this occurs in the coast. Thus, we suggest the occurrence of sympatric study, probably due to their wider altitudinal distribution ranges, lineages at least in Fray Jorge National Park, corresponding to sub- where the Andes would not constitute a barrier to gene flow species P. g. minor (described in literature from coast zones) and P. between populations. g. gayi (detected in this study). The absence of coincidence of phylogeographic patterns found Although Geneland results with P. plebejus showed a similar in co-distributed Phrygilus species suggests that these Andean pattern to that of P. gayi, in which the Chilean Altiplano population birds have not responded congruently to the same historical pro- was isolated from the rest of localities in the absence of geograph- cess. Alternatively, it is possible that these species have not been ical barriers, the MJN did not show clearly differentiated hap- co-distributed for a sufficiently long time to show similar evolu- logroups in this species. This indicates that the biogeographic tionary trajectories. Factors associated with natural selection break found in the Chilean Altiplano could be attributed to the and/or vicariant events may have driven these varied evolutionary small sample size (low haplotype frequencies) and if we increase patterns in the Phrygilus species studied (Bonaccorso, 2009; the number of samples in the studied localities we would probably García-Moreno et al., 2004; Milá et al., 2009; Victoriano et al., detect a single genetic group as observed in the MJN. Similarly in P. 2008). Recent studies have found variable phylogeographic pat- fruticeti, Geneland results showed two distinct geographical terns in co-distributed avian species that have been attributed to R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163 161 natural selection. Zhang et al. (2012) found contrasting evolution- semi-arid scrub plants dominated (Heusser, 1990; Villagrán and ary responses to the same historical process in two co-distributed Varela, 1990; Villagrán, 1993). The expansion signature detected corvid species of China, which were attributed to the differences in in P. alaudinus at Atacama Desert could be related to these climatic habitat preference and breeding behavior. Similarly, Qu and Lei and vegetation variations associated mainly with long-term (2009) observed that two co-distributed passerine birds of the changes in the El Niño Southern Oscillation (ENSO). Tibetan plateau, which share habitats and experienced similar geo- logical and environmental changes, showed differences in their 4.5. Congruence in intraspecific taxonomy from genetic and gene distribution. These differences were attributed to ecological morphological data factors such as differences in dispersal abilities and the specific altitude of the regions where each species inhabits. In the case of A number of studies have shown that broadly distributed avian P. fruticeti and P. plebejus we detected a single genetic group, which species have marked genetic and morphological variation, which is could be attributed to differences in their dispersal capacities in reflected in a great number of described subspecies (Bonaccorso, relation to the rest of Phrygilus species. Moreover, the existence 2009; Bowie et al., 2006; Chaves and Smith, 2011; of sympatric lineages of P. gayi in northern Chile suggests local Gutiérrez-Pinto et al., 2012; Lougheed et al., 2013; Lovette, adaptation to environmental conditions. However, more research 2004). The genus Phrygilus shows a similar pattern; species with on the biology and ecology of these Andean birds is necessary to wide distribution ranges possess great morphological variation, elucidate the effect of natural selection in their evolutionary pat- which is demonstrated by variation in plumage color pattern, body terns. While we detected phylogeographic patterns that could be size and shape and size of beak (Piloni and Camperi, 2011; Fjeldså related to natural selection and local adaptation, our results indi- and Krabbe, 1990; Goodall et al., 1946). Six subspecies have been cated that allopatric events have been a major factor in the popu- described for P. alaudinus and P. unicolor, three for P. fruticeti and lation differentiation of Phrygilus species. Such is the case of P. P. gayi and two subspecies for P. plebejus (Clements et al., 2013). alaudinus, whose evolutionary intraspecific pattern is closed All these species inhabit a broad latitudinal range and are general- related to the Andes; and P. atriceps and P. unicolor, where Lake ists in their altitude range (sea level and also above 3000 m, Titicaca acts as a barrier to gene flow among populations. (Ridgely and Tudor, 1989). P. atriceps, in contrast with the other Phrygilus, has been described as a monotypic highland specialist 4.4. Demographic history species that occurs in a restricted area from the Andes of south- western Peru to western Bolivia, northern Chile and northwestern Our results showed a signature of expansion in three of the six Argentina (Clements et al., 2013; Ridgely and Tudor, 1989). studied species: P. alaudinus and P. fruticeti, which belong to the A variety of studies have demonstrated incongruence in the oldest plumage group (Clade II) dating to the Pliocene (1.8 Myr), delimitation of subspecies through morphological features and and P. plebejus, which belongs to Clade I (1.1 Myr). This is shown molecular data (Cheviron et al., 2005; Fernandes et al., 2013; for the three species by the significant and negative values of Weir et al., 2008). Considering the Chilean localities, our results Tajima’s D and Fu’s Fs, the star-like topology of the MJN and the showed coincidence in intraspecific taxonomy when using mor- BSP. The BSP showed a stronger signature of expansion for P. alaud- phological and molecular data in all cases except for P. gayi. inus (Cluster A), less for P. fruticeti (single genetic group) and a Moreover, for P. fruticeti our study highlights an incongruence with slight expansion for P. plebejus (single genetic group). In P. alaudi- the assignation of subspecies based on the literature. Although the nus the expansion signature was detected about 15,000 years ago literature describes the presence of two subspecies in the study in the lowlands, while P. fruticeti and P. plebejus showed the expan- area (P. f. coracinus in the highlands and P. f. fruticeti in the low- sion signature about 60,000 years ago in the highlands. lands of Chile), our morphological and genetic data suggested the Placzek et al. (2013) performed a reconstruction of past climate occurrence of only a single subspecies. In Chile, P. alaudinus, P. atri- and hydrologic variation of large lakes in the Altiplano from shore- ceps and P. plebejus were represented by a single haplogroup and line deposits and sediment cores over the last 130,000 years. They morphological group, which were coincident with the only sub- found a strong correspondence between these records with periods species described in the study area. P. gayi showed two divergent of higher lake levels between 122,000 and 80,000 years ago. haplogroups, corresponding to subspecies P. g. minor and P. g. gayi, Moreover their modeling efforts underscore the relative aridity of which is concordant with the intraspecific assignation based on the the Altiplano during the global cold interval between 80,000 and literature using phenotypic characters. The lack of morphological 20,000 years ago, when rare and small lake cycles produced smal- differences between subspecies could be due to the small sample ler paleolakes of intermittent temporal presence (Placzek et al., size of Llanos de Challe (n = 2). These findings highlight the impor- 2013). The onset of population expansion in P. fruticeti and P. ple- tance of performing more research integrating molecular and phe- bejus in the highlands occurred about 60,000 years ago, which is notypic data to elucidate the taxonomic status of these species, concordant with this dry period that started about 80,000 years particularly P. gayi and P. fruticeti. ago. Afterwards expansion of paleolakes in the dry Altiplano condi- tions could have been auspicious for re-colonization of these 4.6. Intraspecific taxonomy and conservation considerations Andean birds. In the case of P. alaudinus the expansion occurred about At the intraspecific level it is possible to define Evolutionarily 15,000 years ago in the lowlands, specifically in the Atacama Significant Units (ESUs), which are reciprocally monophyletic units Desert in northern Chile. Stuut and Lamy (2004), who studied for mtDNA and a primary source of species historic genetic diver- the climate variability in Atacama Desert during the last sity (Moritz, 1994). The identification of ESUs as defined above 120,000 years, observed that before 31,000 years ago, conditions requires information on the distribution and phylogeny of in this zone were relatively dry. After this period, a humid phase mtDNA alleles and on the distribution of nuclear alleles. With commenced, which culminated about 17,000 years ago. our phylogenetic results 2 ESUs can be defined for P. gayi and P. Additionally they detected that from 17,000 to 11,000 years ago a unicolor. For P. gayi we can define Cluster A composed of Llanos new drying phase started, which had similar conditions to the per- del Challe (lowlands) and Cluster B formed by all the rest of local- iod before 31,000 years ago. Similarly, palynological studies have ities from Argentina, northern and central Chile (lowlands and revealed that about 10,000 years ago in northern and central highlands). In the case of P. unicolor, the ESUs defined are Cluster Chile vegetal aquatic taxa decreased and herbaceous and A, composed of Peruvian localities (except Puno, highlands) and 162 R. Álvarez-Varas et al. / Molecular Phylogenetics and Evolution 90 (2015) 150–163
Cluster B formed by Chilean and Argentinian populations (high- Brower, A.V.Z., 1996. Parallel race formation and the evolution of mimicry in lands). In this last species, the BEAST analysis reveals a deep diver- Heliconius butterflies: a phylogenetic hypothesis from mitochondrial DNA sequences. Evolution (N.Y.) 50, 195–221. gence, even older than that of Clade III. This finding suggests that Brumfield, R.T., Capparella, A.P., 1996. Historical diversification of birds in populations to both sides (north–south) from the Titicaca Lake northwestern South America: a molecular perspective on the role of vicariant could be different species, which highlights the need for further events. Evolution (N.Y.) 50, 1607–1624. Burns, K.J., Naoki, K., 2004. Molecular phylogenetics and biogeography of research. The classification of these ESUs in P. atriceps and P. uni- Neotropical tanagers in the genus Tangara. Mol. Phylogenet. Evol. 32, 838–854. color suggests that altitude differences (lowlands and highlands) Cadena, C.D., Klicka, J., Ricklefs, R.E., 2007. Evolutionary differentiation in the are not responsible for population connections and isolation in Neotropical montane region: molecular phylogenetics and phylogeography of Buarremon brush-finches (Aves, Emberizidae). Mol. Phylogenet. Evol. 44, 993– these Andean species as in other species of birds 1016. (Arbeláez-Cortés et al., 2010; Barrera-Guzmán et al., 2012; Cadena, C.D., Gutiérrez-Pinto, N., Dávila, N., Chesser, R.T., 2011. No population Gutiérrez-Pinto et al., 2012). By increasing the sampling related genetic structure in a widespread aquatic songbird from the Neotropics. Mol. Phylogenet. Evol. 58, 540–545. to phenotypic traits and by integrating molecular and ecological Campagna, L., Geale, K., Handford, P., Lijtmaer, D., Tubaro, P.L., Lougheed, S.C., 2011. data an appropriate taxonomic classification at intraspecific level A molecular phylogeny of the Sierra-Finches (Phrygilus, Passeriformes): extreme can be established in order to maintain local evolutionary pro- polyphyly in a group of Andean specialists. Mol. Phylogenet. Evol. 61, 521–533. cesses and the potential for evolutionary changes in the future of Chaves, J.a., Smith, T.B., 2011. Evolutionary patterns of diversification in the Andean hummingbird genus Adelomyia. Mol. Phylogenet. Evol. 60, 207–218. these Andean avian species. Chaves, J.A., Pollinger, J.P., Smith, T.B., LeBuhn, G., 2007. The role of geography and ecology in shaping the phylogeography of the speckled hummingbird (Adelomyia melanogenys) in Ecuador. Mol. Phylogenet. Evol. 43, 795–807. Acknowledgments Cheviron, Z.A., Brumfield, R.T., 2009. Migration-selection balance and local adaptation of mitochondrial haplotypes in rufous-collared sparrows (Zonotrichia capensis) along an elevational gradient. Evolution 63, 1593–1605. Financial support for this study was provided by Fondecyt Cheviron, Z.A., Hackett, S.J., Capparella, A.P., 2005. Complex evolutionary history of a Project N° 1100695 and N° 1130948, and Conicyt Master Neotropical lowland forest bird (Lepidothrix coronata) and its implications for Scholarship. We thank to the team of Daniel González for their con- historical hypotheses of the origin of Neotropical avian diversity. Mol. Phylogenet. Evol. 36, 338–357. tribution in the sampling and Eduardo Palma for helpful comments Clements, J.F., Schulenberg, S.T., Iliff, M.J., Sullivan, B.L., Wood, C.L., Roberson, D., on this manuscript. Finally thanks to Daly Noll, Nicole Sallaberry, 2013. The eBird/Clements checklist of birds of the world: Version 6.8. eBird/ Isidora Mura, Michel Sallaberry, Robert Petitpas, David Morales Clements Checkl. birds world Version 6.8.
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