Multi-locus phylogeny and divergence time estimates of Enallagma damselflies (Odonata: Coenagrionidae)☆
Graphical abstract
Introduction
Accurate estimation of divergence times is essential to understanding many evolutionary processes. Time-calibrated phylogenies are important for answering questions about a variety of topics such as biogeography, estimation of diversification rates, and phenotypic evolution. One particular area of interest is how recent climatic changes have influenced the diversification of taxa. The Earth’s climate has experienced fluctuations throughout its history. The magnitude of these fluctuations on biological diversity is of great interest as we move toward a better understanding of anthropogenic climate change.
Previous research has demonstrated that climatic fluctuations during the Quaternary have left distinct genetic signatures across a variety of biota (Hewitt, 2000, Hewitt, 2004). While there were repeated glacial advances and retreats, impacts from these Pleistocene fluctuations should be more pronounced within the last 0.7 million years, a period of more intense glacial cycles (Webb and Bartlein, 1992). These repeated cycles are thought to have promoted divergence in many taxa (Knowles, 2001, Carstens and Knowles, 2006, Mila et al., 2007, Shepard and Burbrink, 2009, Niemiller et al., 2012). However, few studies have focused on obtaining accurate divergence dates to verify that speciation events coincide with major climatic events.
Estimating time-calibrated phylogenies is notoriously difficult, but recent advances in “relaxed-clock” models have led to more accurate estimates of divergence times (Thorne and Kishino, 2002, Sanderson, 2003, Drummond et al., 2006, Drummond and Rambaut, 2007). Although it has been well known for decades that a gene tree may be different from the species tree, most of these divergence time methods were applied to a single gene tree, or to a concatenated data matrix of multiple genes. Recent advances allow joint estimation of a set of gene trees within a shared species tree using a multispecies coalescent model. A commonly used method, ∗BEAST, uses a fully Bayesian framework to estimate the species tree (Heled and Drummond, 2010). This method allows for estimation of divergence times on the species tree and thus more accurate estimates of divergence times can be obtained.
With 38 described species, Enallagma damselflies (Zygoptera:Coenagrionidae) are one of the most diverse groups of Odonata in North America (Westfall and May, 2006). Previous phylogenetic analyses based on mitochondrial DNA and morphological data have consistently separated this genus into two major clades, the “Northern” and “Southern” clades, indicating where the centers of diversity for each is greatest (Northeastern U.S. versus Southeastern U.S., respectively) (Brown et al., 2000, Turgeon et al., 2005). Using a strict molecular clock with a rate of sequence divergence of 2.3% per million years based on Brower’s insect mtDNA clock (Brower, 1994), the last common ancestor of these two major clades was estimated to be approximately 15 million years ago (Turgeon et al., 2005). The Northern clade is composed of three recent radiations (<250,000 years) whereas the Southern clade is composed of mostly older species. Additionally, the estimated diversification rates increased in the last million years by ∼24x in the Northern clade versus only ∼6× in the Southern clade (Turgeon et al., 2005). These results suggest that the Pleistocene glacial cycles had a profound effect on the diversification of this genus.
In this study we expand on the mtDNA dataset of Turgeon et al. (2005) with nuclear loci and analyze these data with a multispecies coalescent approach to gain more robust estimates of divergence dates. Additionally, we infer species diversification rates through time using the estimated time-calibrated species tree. This will allow for a better understanding of the impact of Quaternary climatic oscillations on the diversification of this genus of damselflies in North America.
Section snippets
Taxon sampling and DNA sequencing
We expanded the taxon sampling from Turgeon et al. (2005) for 20 of the 38 Enallagma species, using adult male individuals. Only adult males were sampled because, unlike females, they are easily identified to species based on the morphologies of their reproductive appendages (Westfall and May, 2006). We also added one species, E. coecum. They were collected and preserved by ourselves or collaborators during the summers of 2008–2010 (Table 1) using either 99% ethanol or desiccation. Genomic DNA
Results
We collected sequences from 421, 279, and 303 individuals from 42, 41, and 42 species for locus COI/COII, EF1a, and ITS, respectively. Our results support the following relationships within Enallagma: (1) four recent radiations (green-A, blue-B, orange-C and red-E) and (2) a monophyletic group of older species (purple-D) (Fig. 1). Previous analyses of mtDNA and morphological characters have revealed strong support for the red-E and purple-D clades to be sister to each other (Brown et al., 2000,
Discussion
This is the first multi-locus estimate of a species tree with divergence dates for a group of damselflies. Our results highlight the importance of Pleistocene glacial cycles on the diversification of this genus of damselflies. This study sheds light on key factors that promote diversification within a group of organisms that can serve as models for important ecological and evolutionary processes. Odonata have been well-studied in terms of ecology and behavior and have been implicated as
Acknowledgments
We thank J. Oaks, L. Symes, M. Holder, R. Calsbeek and N. Dominy for comments on drafts of the manuscript. We also extend thanks to J. Oaks and L. Symes for help in the field and all of the people who sent us damselflies from across North America (listed in Table 1). This work was supported by National Science Foundation grant DEB-0714782 to M. A. M. and funds from Dartmouth College.
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This paper was edited by the Associate Editor S.L. Cameron.