Analysis of DNA sequences of six chloroplast and nuclear genes suggests incongruence, introgression, and incomplete lineage sorting in the evolution of Lespedeza (Fabaceae)
Graphical abstract
Highlights
► Neither subgen. Lespedeza nor subgen. Macrolespedeza in Lespedeza is monophyletic. ► Within Lespedeza, the North American clade is sister to the Asian clade. ► Introgression/incomplete lineage sorting caused conflict shallow-level phylogenies. ► Lespedeza originated in Asia in late Miocene. ► The N American ancestor migrated quickly to N America through Bering land bridge.
Introduction
For more than two decades, chloroplast genes have served as useful markers for the reconstruction of plant phylogenies. The advantages of chloroplast data for retrieving phylogeny are based on favorable properties such as a matrilineal (in angiosperms) or patrilineal (in gymnosperms) mode of inheritance, the possession of a single gene copy, a general lack recombination, and the accessibility to universal primers (Palmer et al., 1988; Clegg and Zurawski, 1992). However, despite the potential power and extensive use of chloroplast DNA variation for recovering plant phylogeny, it is widely recognized that under certain circumstances, a chloroplast gene tree will reflect evolutionary processes other than those of phylogenetic descent (Soltis and Kuzoff, 1995, Hardig et al., 2000, Okuyama et al., 2005). When studying relationships among recently diverged taxa of plant species two such processes are introgression and incomplete lineage sorting (Dorado et al., 1992, Degnan and Salter, 2005, Drábková and Vlček, 2010). In closely related species, both introgression and incomplete lineage sorting can lead to discordance between gene trees and confound phylogenetic inference (Rieseberg and Brunsfeld, 1992, Drábková and Vlček, 2010). Thus, recognized signatures can provide insights into the evolutionary process which cannot be detected from other data such as ancient hybridization.
The genus Lespedeza Michx. (Fabaceae: Papilionoideae), with approximately 30 well-known natural hybrids (Clewell, 1966, Akiyama and Ohba, 1982, Ohashi, 2005), provides an ideal system for studying hybridization, introgression, and incomplete lineage sorting. Lespedeza belongs to subtribe Lespedezinae of the tribe Desmodieae. It comprises 40 herbaceous and shrubby species dispersed throughout East Asia and eastern North America, and introduced from Malesia to NE Australia (Nemoto et al., 1995, Ohashi, 2005, Huang et al., 2010, Xu et al., submitted for publication-a). Lespedeza is normally divided into two subgenera, i.e., subgen. Lespedeza and subgen. Macrolespedeza (Maxim.) Ohashi (Ohashi, 1982a, Huang et al., 2010). Morphologically, species of subgen. Macrolespedeza are shrubs with chasmogamous flowers while those of subgen. Lespedeza are herbs or subshrubs with both cleistogamous and chasmogamous flowers. Geographically, species of subgen. Lespedeza occur disjunctly in eastern North America and East Asia, while those of subgen. Macrolespedeza are limited to East Asia.
Campylotropis Bunge and Kummerowia Schindl. are often considered to be indistinct from Lespedeza because they are very similar morphologically. Ohashi (1971) recognized Lespedeza and Kummerowia as distinct genera and Campylotropis as congeneric with Lespedeza. Later, he regarded Kummerowia as congeneric with Lespedeza, while treating Campylotropis as a distinct genus (Ohashi, 1982b). Floral morphology, inflorescence structure, and cpDNA (RFLPs of chloroplast DNA) evidence demonstrate that Lespedeza is more closely related to Kummerowia than to Campylotropis (Nemoto et al., 1995). The pollen morphology indicates that Lespedeza is more closely aligned with Kummerowia and Campylotropis though all three taxa are distinct (Xu et al., 2011). Phylogenetic analysis based on EST-SSR markers reveals Kummerowia to be non-monophyletic. Two accessions of the genus sampled are embedded within a paraphyletic Lespedeza (Wang et al., 2009). Recent phylogenetic analyses based on DNA sequences of chloroplast and nuclear genes of 7–36 accessions of Lespedeza separates Campylotropis from Kummerowia and Lespedeza, while the latter two genera remain unresolved (Stefanović et al., 2009, Nemoto et al., 2010, Han et al., 2010).
In a recent study with 39 out of 40 species of Lespedeza sampled, the two-subgenus classification based on macro-morphology is supported by pollen morphology (Xu et al., 2011). Using restriction fragment length polymorphisms (RFLPs) of cpDNA of 12 accessions of Lespedeza, Nemoto (1995) found that subgen. Lespedeza is not monophyletic. Based on ITS sequences of 10 species of Lespedeza from China and ISSR data of nine species, Zhao (2006) found subgen. Macrolespedeza is monophyletic while subgen. Lespedeza is not. Using macro-morphological data, pollen characters, and chromosome numbers of 12 taxa (nine species, one subspecies, and two varieties) of Lespedeza from Inner Mongolia, Hong (2003) concluded that both subgen. Macrolespedeza and subgen. Lespedeza are monophyletic. Most recently, Nemoto et al. (2010) and Han et al. (2010) used nuclear and chloroplast gene sequences to suggest that subgen. Lespedeza was paraphyletic in relation to the monophyletic subgen. Macrolespedeza. However, two intermediate species, L. dunnii Schindl. and L. fordii Schindl., assigned to subgen. Macrolespedeza, were not sampled in their studies. Furthermore, their sampling size is relatively small with only 35 or 36 accessions representing the 35 species included.
By examining DNA sequence variation of five chloroplast and one nuclear region of 101 samples representing all 40 species recognized in a recent revision (Xu et al., submitted for publication-a), the present study focuses on the following questions: (1) What are the phylogenetic relationships among Lespedeza and its allied genera Campylotropis and Kummerowia? (2) What are the phylogenetic patterns of divergence at the intrageneric level? (3) Can complete species sampling and dense population sampling help resolve the relationships among species? (4) What impact did introgression and incomplete lineage sorting play in the species-level evolution in Lespedeza? (5) What are the divergence times within Lespedeza and among related genera? (6) How did Lespedeza acquire today’s intercontinental disjunction?
Section snippets
Taxon sampling
Forty species, including Lespedeza jiangxiensis Bo Xu, X.F. Gao and L.B. Zhang, a newly described species (Xu et al., submitted for publication-b), were included in the study. Both species of Kummerowia and three species of Campylotropis (37 species in temperate Asia; Iokawa and Ohashi, 2002), i.e., C. delavayi (Franch.) Schindl., C. hirtella (Franch.) Schindl., and C. macrocarpa (Bunge) Rehd. were included as outgroups. Because the relationships of Lespedeza with Campylotropis and Kummerowia
Results
Results of the partition homogeneity test for combined chloroplast and ITS sequences reveals that the data sets are significantly different (p = 0.001) from random pairwise partitions of the data. Therefore, we did not draw any major conclusions based on the analysis using combined chloroplast and ITS data. We conducted a combined analysis with some species/accessions excluded only to test the robustness of the sister relationship between Lespedeza and Kummerowia.
Extrageneric relationships and the monophyly of Lespedeza
There are several basic morphological features that distinguish Campylotropis and Kummerowia from Lespedeza though Lespedeza is more closely related to Kummerowia than to Campylotropis. The inflorescence has often been used as an important feature to differentiate the three genera (Maximowicz, 1873, Schindler, 1913; Nakai, 1939; Ohashi et al., 1981, Nemoto et al., 1995). The inflorescences of Kummerowia are reduced compound cymes which are different from the racemose inflorescences of
Acknowledgments
We thank the herbaria KUN, MO, and PE for providing samples and two anonymous reviewers and Jacqueline A. Van De Veire for their constructive comments.
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