An Examination of Genomic and Acoustic Differentiation Between Eastern, Lilian’S, and Western Meadowlarks (Sturnella Magna, S
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An examination of genomic and acoustic differentiation between Eastern, Lilian’s, and Western Meadowlarks (Sturnella magna, S. m. lilianae, and S. neglecta) By Johanna Beam Ecology and Evolutionary Biology, University of Colorado Boulder Defense Date: March 17th, 2020 Thesis Advisor Scott Taylor, Ecology and Evolutionary Biology Defense Committee Scott Taylor, Ecology and Evolutionary Biology Pieter Johnson, Ecology and Evolutionary Biology Nathan Pieplow, Program for Writing and Rhetoric Table of Contents Abstract……………………………………………………………………………….…………..2 Introduction…………………………………………………..…………………………………..3 Song Methods…………………………………………………..………………………………...7 Genomic Methods…………………………………………………..……………………………9 Song Results….……….………………………………………..……………………………..…11 Genomic Results……………………………………………..………………………………….12 Discussion……………………………………………..………………………………………...13 Taxonomic Implications………………………………………………………………………..16 Conclusions……………………………………………………………………………………...17 Acknowledgements…………………………………………….……………………………….17 Literature Cited…………………………………………….…….…………………………….19 Figures…………………………………………………………………………………………...28 ABSTRACT Understanding species boundaries is a fundamental, but challenging, component of describing and understanding the generation and maintenance of biodiversity. Examining differences among very recently diverged populations can provide insight into the traits and evolutionary mechanisms that drive divergence. The genus Sturnella includes two recently diverged species, the Eastern (Sturnella magna) and Western (S. neglecta) Meadowlark, the former of which has a complex of subspecies distributed across the Americas. Of the Eastern Meadowlark subspecies that occur in the U.S., S. m. lilianae is the only one with a disjunct range in the southwestern U.S. and central Mexico. It also has markedly different song patterns than all other Eastern Meadowlark subspecies. In order to assess population differentiation, we performed whole genome sequencing of 35 birds as well as analysis of various song characteristics, including maximum and minimum frequencies. Results were visualized using principal component analyses and analyzed by running linear discriminant function analyses. S. m. lilianae exhibits high levels of genetic and vocal differentiation from both the Eastern Meadowlark and the Western Meadowlark, and likely forms a distinct evolutionary lineage. Additionally, the subspecies S. m. auropectoralis shows no genetic or acoustic differentiation from S. m. lilianae, suggesting that the subspecies falls within the lilianae group and not the magna group. INTRODUCTION Understanding species boundaries is a fundamental component of describing and understanding the generation and maintenance of biodiversity (Coyne and Orr 2018). From a conservation perspective, it is important to accurately characterize existing biodiversity to inform policy (Sites and Crandall 1997). While necessary, characterizing species boundaries and delineating species is inherently challenging (Harrison and Larson 2014, Balakrishnan 2005, Sites and Crandall 1997). This comes, in part, from the fact that species complexes can exhibit varying levels of genetic and phenotypic divergence. Recently diverged species that are reproductively isolated may share allelic variants because little time has passed for the accumulation of genomic differences due to genetic drift or selection (Campagna et al. 2017). Alternatively, interbreeding (i.e., hybridization) between divergent species may homogenize their genomes and make understanding their evolutionary history and status as species more complicated (Toews et al. 2016c). Examining differences, both genetic and phenotypic, among recently diverged populations can provide insight into the traits and evolutionary mechanisms that drive or maintain divergence between species. Birds exhibit considerable variation in both color and acoustic traits across species. Variation in color and song has variously been linked to species boundaries in birds (Toews and Irwin 2008, Irwin et al. 2018), either their generation (e.g., Toews and Irwin 2008) or their maintenance (e.g., Seddon 2005). Bird song and plumage act as vectors of communication between individuals, conveying important information such as sex, species, and fitness (Catchpole and Slater 1995, Stein and Uy 2006). Female mate choice based on song and plumage has important implications for the maintenance or erosion of species barriers: if a female chooses a mate from the wrong species, the offspring produced may be unviable, infertile, or have lower fitness, all of which would lead to the maintenance of species boundaries (Collins 2004, Lanyon 1979, Baker and Boylan 1999, Price and Bouvier 2002). Alternatively, if post zygotic isolation is weak, admixed offspring may survive and reproduce leading to gene flow between species and the erosion of species boundaries. In oscine passerines (passerines that learn their song), geographical variation in song is common and often coincides with genetic variation (Price 2008, Baker and Boylan 1999, Toews and Irwin 2008). In Greenish Warblers (Phylloscopus trochiloides), for example, the subspecies occurring at higher latitudes sing more complex songs, are the most genetically distinct from one another, and rarely hybridize (Irwin 2000, Alcaide 2014). This genetic and acoustic variation may be indirectly caused by ecological differences between the diverging populations across a north- south gradient (Irwin 2000). Because songbirds learn songs not only from their parents, but also from their non-related conspecific neighbors within a population, the possibility for errors to arise is high (Price, 2008). This error, called cultural mutation, can facilitate population divergence in concert with geographical variation, differential selection, and drift when populations have broad geographic distributions or are small and isolated (Price 2008, Irwin et al. 2018). Some songbirds show preferential conspecific song learning (i.e., what songs birds learn to sing), which counteracts cultural mutation and can facilitate the maintenance of species boundaries (Price 2008, Nelson 2000). This appears to be the case in both Swamp Sparrows (Melospiza georgiana) and White-crowned Sparrows (Zonotrichia leucophrys), which do not hybridize readily in nature. In tests of song learning, both species preferentially learned conspecific or con-subspecific song rather than the songs of a closely related heterospecific (Nelson 2000, Marler and Peters 1977). Preferential song learning was examined in social isolation from other conspecifics, suggesting a genetic component to song learning as well as the social component of learning from neighboring birds (Nelson 2000). Presumably, a failure to learn the correct song, failure to interpret the quality of a mate using song, or lack of geographical variation in song between populations could lead to gene flow and the erosion of species boundaries between closely related species. With increasing frequency, detailed analyses of song between closely related species are providing insight into species boundaries (Baker and Logue 2003; Mason et al. 2014). The analysis of song variation in combination with high resolution genomic data can help clarify species boundaries between taxa whose taxonomy has historically controversial (Baker and Boylan 1999, Qvarnström et al. 2010). The genus Sturnella includes two recently diverged species, the Eastern (Sturnella magna) and Western (Sturnella neglecta) meadowlark: S. magna includes a large complex of subspecies that range from South America to the midwestern United States. Of the subspecies of Eastern meadowlark that occur in North America, Lilian’s Eastern meadowlark (Sturnella magna lilianae) and the Cuban Meadowlark (S. m. hippocrepis) are the only subspecies with breeding ranges that are geographically disjunct from the rest of the range of the Eastern Meadowlark (Fink et al. 2020, Arnold 2020, eBird 2020). Lilian’s meadowlark occurs in the desert lowlands of SE Arizona, New Mexico, west Texas, and occasionally into Colorado as demonstrated by Leukering & Pieplow (2009) and may represent a divergent population (Figure 1A). The subspecies lilianae is noted to have separate breeding grounds and different habitat preferences than the Western Meadowlark (S. neglecta), despite range overlap in Arizona and New Mexico (Lanyon W.E. 1962) (Figure 1B). Most recently, Barker et al. (2008) examined two mitochondrial genes (cytochrome b, and ND2), as well as the sex-linked intron ACO1-I9, and presented evidence that the subspecies lilianae, including S. m. auropectoralis, represent a distinct species given their deep genetic divergence from the other S. magna subspecies in the complex. In meadowlarks, song is used both to defend territories and in mate selection (Ordal 1974). Lanyon (1957) observed that both species of meadowlark defend their territories equally against conspecific and heterospecific meadowlarks. Rohwer (1972, 1973) also found that there is divergence in song between Lilian’s and Western taxa in areas of sympatry as well as Eastern and Western taxa in areas of sympatry, signifying that song differentiation persists when meadowlark species co-occur. The call notes of Eastern and Western meadowlarks are likely innate (i.e., genetically inherited) are have distinctive features: Eastern meadowlarks have a high frequency “dzert” call note, and Western have a lower frequency “chupp” call note (Lanyon 1962, Rohwer 1972). The differences in call note structure and frequency are