Detecting reticulate relationships among diploid Leucanthemum Mill. (Compositae, Anthemideae) taxa using multilocus species tree reconstruction methods and AFLP fingerprinting☆
Graphical abstract
Introduction
It is hardly possible to overestimate the importance of hybridisation in plant evolution and to elude the argumentation of Oberprieler (2014) that Biology’s First Law (McShea and Brandon, 2010) saying that “in the absence of selection and constraint, complexity – in the sense of differentiation among parts – will tend to increase” should be augmented by a second principle (and maybe Biology’s Second Law) that complexity does not only increase through differentiation and divergence alone but also through genetic exchange, (re)combination, and phylogenetic reticulation. We are presently experiencing a shift in perspective from the view on hybridisation as a merely destructive process that could lead to a reversal of differentiation and a loss of biodiversity toward an enforced appreciation of hybridisation as a constructive and even creative process in evolutionary biology (Abbott et al., 2013, Yakimowski and Rieseberg, 2014). With an estimated frequency of at least 25% of species that hybridise with each other (Mallet, 2005), the plant kingdom represents a well-suited domain of life for studying this paramount evolutionary process.
While the study of hybridisation processes as “collision of species” finds its analogue in the particle colliders in physics, which allow the study of the internal structure of the components of matter (Buerkle and Lexer, 2008), methods of phylogenetic reconstruction based on molecular evidence could be seen as our instruments equivalent to telescopes in astrophysics that allow us to view back into time and the evolutionary history of an organism group. However, as in physics, the two approaches are connected and microevolutionary processes like speciation and hybridisation events leave their footprint in the evolution of genomic markers that are used in turn to infer the macroevolutionary patterns of phylogenetic relationships. Two natural processes are especially noteworthy that could lead to a blurring of phylogenetic patterns among lineages (species trees) as reconstructed from underlying (and often discordant) evolutionary histories of individual molecular markers (gene trees): incomplete lineage sorting (ILS; Hudson, 1983, Tajima, 1983, Takahata, 1995, Rannala and Yang, 2008) and gene flow among lineages (Slatkin and Maddison, 1989).
Since ‘total evidence’ approaches with concatenated sequence data from markers with high levels of discordance may produce robust and well-supported, but inaccurate phylogenetic reconstructions (Kubatko and Degnan, 2007, Weisrock et al., 2012), an increasing number of methods have been proposed to estimate the correct species tree without concatenation of sequence data, especially for those cases in which ILS is the reason for incongruence among gene trees (Maddison and Knowles, 2006, Mossel and Roch, 2010, Liu, 2008, Than and Nakhleh, 2009, Liu et al., 2009, Heled and Drummond, 2010, Knowles and Kubatko, 2010, Fan and Kubatko, 2011, Leaché and Rannala, 2011, Camargo et al., 2012). Despite continuous efforts to find methods that distinguish between the effects of ILS and hybridisation (Sang and Zhong, 2000, Holland et al., 2008, Maureira-Butler et al., 2008, Joly et al., 2009, Kubatko, 2009, Kelly et al., 2010, Gerard et al., 2011, Blanco-Pastor et al., 2012, De Villiers et al., 2013, Ramadugu et al., 2013) species tree inference jointly considering the two processes remains a great challenge (Leaché et al., 2014) and constitutes a very active field in present phylogenetic systematics; especially in the light of increasing simplifications in the process of gaining huge amounts of sequence data for gene tree reconstructions through next-generation sequencing techniques (Glenn, 2011).
The genus Leucanthemum Mill. (Compositae, Anthemideae) is a large polyploid complex comprising 42 species (The Euro+Med Plantbase, 2014) with ploidy levels ranging from diploid (2x) to dodecaploid (12x), and one species [L. lacustre (Brot.) Samp.) from Portugal] even showing a chromosome number of 2n = 22x = 198 (docosaploid level). The genus is distributed all over the European continent, with one species (L. ircutianum DC.) reaching Siberia and some species introduced to many temperate regions of the northern and southern hemisphere (Meusel and Jäger, 1992). While the reticulate evolutionary history of the genus caused by allopolyploidy was demonstrated in a number of studies based on molecular data (Oberprieler et al., 2011b, Oberprieler et al., 2014, Greiner et al., 2012, Greiner et al., 2013), results obtained in the course of a recent study of sequence variation at the external transcribed spacer region of the nuclear ribosomal repeat (nrDNA ETS) in diploid representatives of the genus suggested either gene flow among or a homoploid hybrid origin of some of these species (Oberprieler et al., 2014): ETS ribotypes realized in Leucanthemum diploids were found to fall into two clusters, a plesiomorphic ETS ribotype cluster closely related to ETS ribotypes of outgroup genera (called the ‘green’ ETS ribotype cluster in Oberprieler et al., 2014) and an apomorphic cluster (‘red’ ETS ribotype cluster). While some diploid species were found being fixed for either of the two types, others exhibited an additive pattern of the two types, which was interpreted as being due to hybridisation and gene flow among diploids in the former study (Oberprieler et al., 2014).
In the present contribution, we use AFLP fingerprinting and species tree inference based on nine nuclear and one plastid gene trees to test the hypothesis that the evolutionary history of Leucanthemum diploids was influenced by gene flow caused by hybridisation or even homoploid hybrid speciation. We were especially interested in finding ways to disentangle effects of incomplete lineage sorting and hybridisation as causes for incongruence among multilocus gene trees in order to quantify the amount of incongruence caused by hybridisation alone and to pinpoint potential hybrid lineages/taxa that exceedingly contribute to a hybrid signal in the data set. In contrast to a method used by Maureira-Butler et al., 2008, Blanco-Pastor et al., 2012, Ramadugu et al., 2013 to detect potential hybrids by examining the effect of sequential taxon deletion on gene tree incongruence, we infer taxon-specific hybrid index scores by a new method based on triplet-permutations and the computation of likelihood scores for the resulting three-taxon species trees or networks following calculations described in Yu et al. (2012). Significance of the taxon-specific hybrid index scores (resulting from the joint effects of incomplete lineage and hybridisation) is then tested by comparing them with the corresponding values from coalescence simulations done under the assumption of the presence of incomplete lineage sorting alone.
Section snippets
Taxon sampling and DNA extraction
Either silica-gel dried plant material collected in the field or herbarium specimens were used in the present study (Table 1, Fig. 1). The 19 diploid Leucanthemum taxa included were represented by 39 accessions; mostly we sampled two accessions per taxon, except in the cases of L. rotundifolium (Willd.) DC. (three accessions), L. vulgare L. subsp. vulgare (three accessions), and L. ligusticum Marchetti et al. (one accession). For the sequence-based analyses, ten representatives of genera
AFLP fingerprinting
AFLP fingerprinting of 39 individuals and nine replicates with three selective primer pairs (E-ACC/M-CTAG, E-AGG/M-CTAG, E-ACA/M-CTAG) and an optimised, automated band-scoring (Holland et al., 2008) yielded 610 polymorphic loci (210, 183, and 217, respectively) in the range of 50–420 bp. An average (Euclidian) error rate of 10% was estimated among all replicates, being a reasonable rate for an automated band scoring procedure (i.e., 6–13% or 9–18% given in Holland et al., 2008). The resolution
Disentangling hybridisation and incomplete lineage sorting (ILS)
A simulation study by Leaché et al. (2014) impressively demonstrated that gene flow could have a considerable influence on species tree estimation and could bias the estimation of the species tree topology and of parameters estimated such as population sizes and divergence times. However, while species tree reconstruction methods usually account for incomplete lineage sorting (ILS) due to its nature as a process intrinsically linked with speciation events (Edwards, 2009), gene flow as the other
Acknowledgments
We would like to thank Dr. Sarah Diermeier (Regensburg; Cold Spring Harbor, NY, USA) for her help with the analysis of NGS results with the Galaxy webportal and Dr. Santiago Ortíz and Dr. Juan Rodríguez Oubiña (both Santiago de Compostela, Spain) for their support on excursions to Galicia (NW Spain). For nomenclatural advice we owe gratitude to Nick Turland (Berlin). The technical help of Mr. Peter Hummel in the molecular laboratory of the working group at the University of Regensburg is
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This paper was edited by the Associate Editor Xiao-Quan Wang.