Genetic divergence analysis of the Common Barn Owl Tyto alba (Scopoli, 1769) and the Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile using COI sequence

Abstract In this paper new mitochondrial COI sequences of Common Barn Owl Tyto alba (Scopoli, 1769) and Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile are reported and compared with sequences from other parts of the World. The intraspecific genetic divergence (mean p-distance) was 4.6 to 5.5% for the Common Barn Owl in comparison with specimens from northern Europe and Australasia and 3.1% for the Short-eared Owl with respect to samples from north America, northern Europe and northern Asia. Phylogenetic analyses revealed three distinctive groups for the Common Barn Owl: (i) South America (Chile and Argentina) plus Central and North America, (ii) northern Europe and (iii) Australasia, and two distinctive groups for the Short-eared Owl: (i) South America (Chile and Argentina) and (ii) north America plus northern Europe and northern Asia. The level of genetic divergence observed in both species exceeds the upper limit of intraspecific comparisons reported previously for Strigiformes. Therefore, this suggests that further research is needed to assess the taxonomic status, particularly for the Chilean populations that, to date, have been identified as belonging to these species through traditional taxonomy.


Introduction
The Common Barn Owl Tyto alba (Scopoli, 1769) (Strigiformes, Tytonidae) and the Short-eared Owl Asio flammeus (Pontoppidan, 1763) (Strigiformes, Strigidae) are nocturnal owls usually found in open habitats such as farmland and grassland associated with humanized areas (Weick 2006). The range of both species extends across several continents. In Chile, these owls are sympatric over most of their geographic range, and are considered opportunistic generalists, feeding mainly on small mammals (Rau et al. 1992, Martínez et al. 1998, Figueroa et al. 2009). Although the Common Barn Owl and the Short-eared Owl have been widely studied throughout most of their geographic range in Chile, studies have concentrated mainly on ecological aspects, e.g. diet, conservation and habitat (Raimilla et al. 2012). In contrast, their levels of genetic divergence in comparison with populations in other geographic ranges, have received little attention. This is especially important given that Chilean populations of both species are peripheral populations in the southern hemisphere whose geographic isolation, as a consequence of the Andean mountains that act as a geographic barrier, may have led to evolutionary divergence.
The Common Barn Owl includes several subspecies, some of which may represent distinct and endemic species. To date, up to 25 distinct species have been recognized within the Tyto complex but the status of many of these forms remains poorly documented (Weick 2006). Based on morphological data, the Common Barn Owl in Chile has been assigned to the subspecies T. a. tuidara (J.E. Gray, 1829) (Figueroa et al. 2014). In the Short-eared Owl, nine subspecies have been recognized across its geographic range (Weick 2006), of which the subspecies A. f. suinda (Vieillot, 1817) occurs in Chile (Figueroa et al. 2014). Nevertheless, no molecular data supporting the taxonomic identity of the populations of these species in Chile are available yet.
The mitochondrial cytochrome c oxidase subunit I (COI) gene is considered a powerful molecular tool for molecular identification ('barcoding') of bird species and may also help to clarify taxonomic relationships (Hebert et al. 2004, Kerr et al. 2007, 2009, Aliabadian et al. 2009). This gene includes a 648-bp region of the mitochondrial genome that provides reasonably good resolution for identifying animal species, as shown by the fact that deep sequence divergences are found between most closely related pairs of animal species (Hebert et al. 2003). Given this property, i.e. its ability to capture species boundaries, this marker has been widely used as a method for identifying species, using a library of sequences linked to taxonomically verified voucher specimens (Ratnasingham and Hebert 2007).
In this study, we sequenced a 648-bp region of the COI gene of the Common Barn Owl and the Short-eared Owl specimens from southern Chile with the aim of comparing these data with the COI sequences published for these species from other geographic areas. Given that no major information on genetic divergence of owls from Chile based on the COI gene is available, this study may contribute to clarifying the taxonomic status and evolutionary divergence at regional and global scales.

Samples collection and DNA extraction
Owl samples (Common Barn Owl, n = 9; and Short-eared Owl, n = 1) were collected in 2012 and 2013 from dead birds found along the highways (Ruta 5, Ruta 215 and Ruta 207) that connect Osorno with neighboring cities, which run through the provinces of Valdivia, Ranco and Osorno in southern Chile (40°-41° S latitude) (Figure 1). Photographs illustrating the plumage and overall external appearance of Common Barn Owls and Short-eared Owls collected in southern Chile are provided in the Suppl. material 1. DNA was extracted from fixed muscle using the phenol-chloroform method, as described in Taggart et al. (1992). Extracts were standardized at 100 ng/µL using Tris-EDTA buffer pH 8.0.

Phylogenetic and sequence divergence analyses
Summary statistics, variable and parsimony informative sites, and p-distances were calculated with MEGA 5.05 (Tamura et al. 2011). Relationships among COI haplotypes were reconstructed based on the median joining network implemented in the Network program ver. 4.6.1 (Bandelt et al. 1999). Phylogenetic analyses were carried out using Maximum Likelihood (ML) and Bayesian Inference (BI) approaches. The best-fit nucleotide substitution model was selected using Akaike's information criterion (AIC). The best model was then used with the ML analyses to construct a ML tree using MEGA 5.05. The consistency of topologies (nodal support) was estimated using a bootstrap approach with 1000 bootstrap replications (Felsenstein 1985). A Bayesian tree was determined using MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001). The analysis was performed using two simultaneous runs of 1,000,000 generations, a random starting tree with four independent Markov chains (MCMC), and tree sampling every 500 generations. The evolutionary model selected for BI analysis was the GTR + Γ + I. The first 25% of the generations were discarded as burn-in, and posterior probabilities were determined by constructing a 50% majority rule consensus for the remaining trees. The trees were visualized using FigTree ver. 1.3.1 (Rambaut 2009).

Sequence divergence and phylogenetic relationship of common Barn Owl
The sequence alignment of the COI gene obtained had 582 positions in nine specimens of Common Barn Owl from southern Chile, and 12 representative sequences from other regions (one from Argentina, five from North America, two from Central America, two from northern Europe and two from Australasia) obtained from Genbank (http://www.ncbi.nlm.nih.gov/Genbank) (Table 1). For this alignment, five haplotypes, defined by 47 variable positions, were found ( Figure 2a). In this align-    Five haplotypes for the Common Barn Owl specimens were observed, considering all studied localities, with haplotype 1 being exclusive to the New World (including Chile), and representing more than half of the individuals in the data set (61.9%) (Figure 3a). The remaining haplotypes were mostly restricted to a single locality, as were haplotypes 3, 4 and 5, of North America, northern Europe and Australasia, respectively. Of note is that haplotype 2 was exclusive to Chile.
The nine sequences of Common Barn Owl from Chile showed low levels of sequence divergence, ranging from 0 to 0.2% uncorrected p-value. Similarly, genetic divergence between Common Barn Owls from Chile and those from elsewhere in the New World was low, ranging from 0.0 to 0.2%. In contrast, genetic divergence between Common Barn Owls from Chile and those from northern Europe and Australasia was much higher, with p-values ranging from 4.6 to 5.5%. The ML tree showed a cluster of sequences from the New World (including Chile), with a clear separation of sequences from northern Europe and Australasia; this tree had nodes with high (100%) bootstrap values (Figure 4a). The BI tree showed a concordant topology, exhibiting high (0.99-1.00) posterior probability values for each node (Figure 4b).

Sequence divergence and phylogenetic relationship of Short-eared Owl
The sequence alignment of the COI gene for Short-eared Owl had 611 positions in one specimen from Chile, and nine representative specimens from other regions (one from Argentina, four from North America, two from northern Europe and two from northern Asia) (Table 1). Three haplotypes, defined by 20 variable positions, were identified ( Figure 2b). Moreover, in this alignment, 19 positions were parsimony informative, and 17 out of 20 changes were synonymous. The best fit-model of nucleotide substitution was Tamura-Nei (TN93) with 1871.5 Akaike value and parameter estimates for base frequencies of A = 0.2594, T = 0.2507, C = 0.3401 and G = 0.1498, relative substitution rates of AC = 0.0083, AG = 0.2110, AT = 0.0061, CG = 0.0036 and CT = 0.1591, and proportion of invariables sites of 0.9673.
Three haplotypes were found, with haplotype 1 (n = 2 individuals) being exclusive to Chile and Argentina, haplotype 2 (n = 5) exclusive to North America, northern Europe and northern Asia and haplotype 3 (n = 1) exclusive to northern Europe.
COI sequences from Chile and Argentina were identical. However, sequences from Chile/Argentina were highly divergent (3.1%) from those from North America, northern Europe and northern Asia. The ML and BI trees (results not shown) showed one cluster for specimens from Chile and Argentina and another cluster for specimens from North America, northern Europe and northern Asia.

Discussion
The three major groups of Common Barn Owls inferred here by means of phylogenetic analysis of COI sequences (South America plus North America, Australasia and northern Europe) are similar to those identified in previous phylogenetic studies using other molecular markers (mitochondrial cyt b and nuclear LDHb) (Wink et al. 2004, Alaie andAliabadian 2012). In particular, these studies observed a consistent separation between the New World and Old World Common Barn Owls. Moreover, this topology is in accordance with a recent study that used the COI sequence (Nijman and Aliabadian 2013), where strong evidence of a major divergence between New World (furcata clade) and Old World (alba clade) clades was found. The combined DNA evidence is consistent with the recognition of New and Old World Common Barn Owls as two different species (Tyto furcata and Tyto alba, respectively) as was originally proposed by König and Weick (2008). However, more extensive studies of morphological, molecular and acoustic divergence among New and Old World populations are highly desirable.
In addition, the genetic distance among the Common Barn Owls from Chile and northern Europe and Australasia, from 4.6 to 5.5%, is not surprising, given that previous studies have also documented high levels of sequence divergence among American and Old World populations of this species, ranging from 5.3 to 7.2% (Alaie andAliabadian 2012, Nijman andAliabadian 2013). Therefore, our results support the view that the Common Barn Owl shows strong genetic differentiation across its geographic range, in particular among populations from distant geographic areas.
With regard to the Short-eared Owl, the intraspecific divergence of 3.1% is in accordance with previous studies that also used the COI gene, which reported a maximum divergence of 3.3% for the species (Nijman and Aliabadian 2013) or a mean distance of 3.2% between a single specimen from Argentina and multiple specimens from North America, Europe and northern Asia (Kerr et al. 2009). The divergence between Short-eared Owls from (southern) South America and those sampled elsewhere is interesting and indicates that the taxonomic status of South American populations requires detailed taxonomic study. Again, this should include multiple lines of evidence, including morphology and acoustics, in addition to molecular data.
The levels of sequence variation observed for the Common Barn Owls and the Short-eared Owl from Chile compared to those from other continents was well above the mean intraspecific distance reported for bird species, which ranges from 0.23% to 0.43% (Aliabadian et al. 2009, Hebert et al. 2004, Kerr et al. 2007, 2009. These values are higher than any other owl species analysed so far using COI sequence data, given that in these it has been reported a value of average distance less than 2% (Nijman and Aliabadian 2013).
In previous studies, high levels of sequence divergence have often been interpreted as a possible taxonomic artifact, i.e. as an indication that more taxonomic study is warranted. For instance, Hebert et al. (2004) found four out of 260 species of North American birds with intraspecific divergence ranging from 3.7 to 7.2%, and Kerr et al. (2009), studying the birds of Argentina, observed 13 out of 389 species with sequence divergences exceeding 2.4%. In birds, disjunct ranges, differences in ecology, non-migratory behaviour and rapid speciation may be important factors associated with this phenomenon (Kerr et al. 2009). In the case of the Common Barn Owl, the existence of geographic barriers to gene flow among populations on different continents is to be expected, and this in combination with its non-migratory or short-distance migratory behaviour (Matics 2003), should contribute to promote the genetic divergence. However, all New World Common Barn Owls studied in this work were very similar given that no major genetic divergences were observed. This result agrees with Nijman and Aliabadian (2013), who found no latitudinal clusters for Common Barn Owl sequences from different places of America (North America, the Caribbean, northern South America and southern South America). Although further analysis with other molecular markers is needed to support this result; even so, it is likely that the scarce genetic variation of the Common Barn Owl registered across America may reflect the existence of a particular process of divergence.
In conclusion this study provides further evidence that the Common Barn Owl and the Short-eared Owl show high levels of intraspecific COI sequence divergence across their geographic range. The level of genetic differentiation of both species collected in South America was above the upper limit of species reported for Strigiformes, suggesting that further studies are needed to evaluate the taxonomic position of these populations. and two reviewers for their efforts that greatly improved the quality and clarity of the work. The English language editing of the manuscript by Susan Angus from the Dirección de Investigación of the Universidad de Los Lagos, is also appreciated.