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United States Department of Agriculture

Agricultural Research Service


Location: National Clonal Germplasm Repository for Citrus

Title: A six nuclear gene phylogeny of Citrus (Rutaceae) taking into account hybridization and lineage sorting

item Ramadugu, Chandrika
item Pfiel, Bernard E
item Keremane, Manjunath
item Lee, Richard
item Maureira-butler, Ivan J
item Roose, Mikeal L

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/29/2013
Publication Date: 7/16/2013
Citation: Ramadugu, C., Pfiel, B., Keremane, M.L., Lee, R.F., Maureira-Butler, I., Roose, M. 2013. A six nuclear gene phylogeny of Citrus (Rutaceae) taking into account hybridization and lineage sorting. PLoS ONE. 8(7) e68410. DOI:10.1371/journal.pone.0068410

Interpretive Summary: Most of the cultivated varieties of Citrus are derived by hybridization of three basic species: citron (Citrus medica), mandarin (C. reticulata) and pummelo (C. maxima). Citrus can reproduce by both sexual (through seed) and asexual means (polyembryony and budding). Because of its peculiar biology, the species demarcation is not clear in Citrus and sexually nonviable cultivars can be propagated clonally. This makes it difficult to predict the relationships between the different citrus cultivars. In order to understand the phylogenetic relationships of the different Citrus cultivars and their close relatives, we did an analysis with sequences generated from six nuclear genes obtained from 33 cultivars. The phylogenetic trees obtained from the six genes show inconsistencies or, incongruence, since the individual genes are predicted to undergo additional changes due to hybridization, incomplete lineage sorting, gene duplication and loss. Lineage sorting is the process of fixation of alleles along species lineage; for example, alleles specific to mandarins will be found only in the line that evolves into mandarins and not in the line that evolves into pummelos. Most of the cultivated citrus varieties are derived by hybridization – both natural and man-made. During the course of evolution, different lineages of citrus acquire distinct characters. At times, these characters may not be as distinct because of incomplete lineage sorting. If the species being studied are derived from an ancestor that existed a long time ago, it is possible that given sufficient time, the distinct characters of a lineage revert back to the ancestral characters. All these factors can result in incomplete lineage sorting. As we go back in time, two divergent lineages “coalesce” if they are derived from a common ancestor. Using coalescence simulation testing methods we tried to understand the cause of incongruence in the gene trees. Previous study done using simple sequence repeat markers (SSR) has indicated the possible hybrid nature of several citrus accessions. We compared our results with the previous published data using SSR markers and used the patterns observed among the six nuclear loci to identify incomplete lineage sorting, hybridization, or other processes in the different accessions studied. We identified hybrids by coalescence simulation testing that could not be detected by other methods, such as SSR markers. The study leads to a better understanding of the phylogenetic relationships in this biologically complex genus.

Technical Abstract: Background: Citrus (Rutaceae) comprises of many important cultivated species which generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4-12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships. Methodology and Principal Findings: 1. We generated phylogenetic trees using haplotype sequences of six nuclear genes and utilized single nucleotide polymorphism data to analyze relationships. 2. Published simple sequence repeat data was re-analyzed to study population structure and the results were compared with phylogenetic trees constructed using sequence data and with the results of coalescence simulations. 3. To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred. These ‘species trees’ have then been used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from the observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test. Conclusions: We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches – single nucleotide polymorphism study, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer phylogeny among species of Citrus.

Last Modified: 06/23/2017
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