Back-and-forth hermaphroditism: phylogenetic context of reproductive system evolution in subdioecious daphne laureola conchita Alonso1,2 and Carlos M. Herrera1

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Conchita Alonso1,2 and Carlos M. Herrera1
1 Estacio´ n Biolo´ gica de Don˜ ana, Consejo Superior de Investigaciones Cientı´ficas (CSIC). Apdo 1056, 41080, Sevilla, Spain
2 E-mail:

Recent phylogenetic analyses of sexual reproductive systems supported the evolutionary pathway from hermaphroditism to dioecy via gynodioecy in different groups of angiosperms. In this study, we explore the evolution of sexual reproductive systems in Daphne laureola L. (Thymelaeaceae), a species with variation in reproductive system among population. Sequences from the ITS region of the nuclear ribosomal cistron and two plastid markers (psbA-trnH and ndhF) were analyzed and used to map the population reproductive system along the molecular phylogeny. Our results support D. laureola as a monophyletic lineage with three different clades within the Iberian Peninsula. The hermaphroditic populations belong to two different clades, whereas gynodioecy is ubiquitous but characteristic of the third clade, which grouped together all the North-Western Iberian populations sampled, including the apparently oldest haplotype sampled. Gynodioecy appears as the most likely basal condition of the 13 analyzed populations, but different evolutionary transitions in reproductive sexual system were traced within each D. laureola clade. Both ecological conditions and (meta)population dynamics may help explain plant reproductive system evolution at the microevolutionary scale. Phylogenetic studies in which the historical relationships between populations differing in reproductive system can be ascertained will help to clarify the process.
KEY W ORDS: Geographic variation, gynodioecy, Iberian Peninsula, ITS, microevolution, molecular markers, phylogeography.

Hermaphroditism is considered the ancestral condition from which all other sexual reproductive systems have evolved in the angiosperms (see Barrett 2002 for a review). Gynodioecy, where hermaphrodite and female individuals coexist within pop- ulations, has been traditionally viewed as one intermediate evo- lutionary stage between the two most common reproductive sys- tems, hermaphroditism and dioecy. The evolutionary pathway from hermaphroditism to dioecy via gynodioecy has been sat- isfactorily modeled theoretically, and requires the spreading of male-sterile mutants into cosexual populations followed by se- lection against female function in the remaining hermaphrodites (Schultz 1994; Charlesworth 1999). At the first stage, to spread

and persist within populations, the male-sterile mutants must have some consistent fecundity advantage that compensates for their gametic disadvantage. In theory, the female frequency within sin- gle populations of infinite size would be regulated by the relative seed production of each sexual morph, and the degree of selfing and inbreeding depression (Charlesworth 1999). Metapopulation models have further indicated that whenever a population is sub- divided into local groups, individual fitness will be also a function of local morph frequency and pollen limitation of seed produc- tion, which would determine if evolutionary processes allow the maintenance of polymorphism (McCauley and Taylor 1997). The stability of gynodioecy can be also favored by nucleo-cytoplasmic

sex determination (Bailey et al. 2003; Bailey and Delph 2007; Dufay et al. 2007; but see Schultz 1994).

In the last two decades important efforts have been made to verify these models, explain some seeming exceptions to the rule, and understand the ecological conditions favoring such mi- croevolutionary transitions in sexual reproductive system (Delph and Carroll 2001; Medrano et al. 2005; Ashman 2006; McCauley and Bailey 2009). In addition, some phylogenetic analyses of reproductive system evolution based on molecular markers (see Weller and Sakai 1999 for a review; Weiblen et al. 2000; Vamosi et al. 2003; Navajas-Pe´rez et al. 2005) have also supported the theoretical pathway for a few plant groups in a macroevolution- ary context, and reversals from dimorphism to hermaphroditism have been also inferred at the interspecific level (e.g., Weller et al.

1995; Desfeux et al. 1996; Weiblen et al. 2000).

Links between the micro and macroevolutionary approaches are being provided by studies of geographical variation in species that exhibit among-population variation in reproductive system (hereafter referred to as subdioecious). These investigations have shown that environmental stress, founder effects, and interactions with pollinators, are commonly correlated with the frequency of females in subdioecious species (e.g., Case and Barrett 2004; Nilsson and A˚ gren 2006; Ramsey et al. 2006; Alonso et al. 2007; Caruso and Case 2007). Finally, intraspecific phylogeographic studies in which the historical relationships between populations differing in reproductive system can be ascertained using molec- ular markers may also provide crucial information to identify the most common transitions in reproductive system evolution at the intraspecific level. By mapping selected characters on a phylogeny, such studies can suggest whether other morpholog- ical, functional, and ecological characters have played a role in this microevolutionary process. However, such studies are scarce (see e.g., Fe´nart et al. 2006), most likely because of the difficul- ties in finding well-supported intraspecific phylogenetic trees and intraspecific variation in reproductive system simultaneously for the same species.

Our study uses a phylogenetic approach to provide a historical frame for the evolutionary transitions between hermaphroditism and gynodioecy at an intraspecific level. Daphne laureola L. (Thymelaeaceae) is a shrub widely distributed in the understory of European forests. Monomorphic (i.e., hermaphroditic) and gynodioecious populations are patchily dis- tributed over the Iberian Peninsula (Alonso et al. 2007). Gyn- odioecious populations prevail in the northwest and southeast of this large geographic area and, most likely, also at other disjunct areas of the species range (gynodioecious populations occur also in Slovenia and Morocco; C. Alonso, unpubl. data). In addition, there are at least two disjoint, distant areas in the northeast and southwest of the Iberian Peninsula in which pure hermaphroditic populations exist. We attempt to understand such a geographic

pattern using molecular phylogenetic tools. Combining different marker systems is usually advantageous to unravel intraspecific evolutionary histories; thus we analyze the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron, and two plastid markers (psbA-trnH and ndhF). Nuclear and plastid mark- ers show contrasting modes of inheritance. It is thus particularly interesting to combine both to track plant reproductive system evolution because, as mentioned above, gynodioecy is frequently determined by the interaction between maternally inherited cy- toplasmic male sterility (CMS) factors and biparentally inher- ited nuclear genes that restore male fertility (Kaul 1988; but see McCauley and Olson 2008).

The following questions are addressed in this article. What are the phylogenetic relationships between D. laureola popula- tions from different Iberian regions and how does the sexual re- productive system change along the molecular phylogeny? Are D. laureola populations from the hermaphroditic disjunct areas genetically more related to each other rather than to gynodioe- cious populations? If so, is hermaphroditism the ancestral trait among D. laureola populations? Or alternatively, in which direc- tion and how many times has the transition between gynodioecy and hermaphroditism occurred?

Materials and Methods


Daphne laureola L. (Thymelaeaceae) is an early-season flower- ing, insect-pollinated and bird-dispersed, evergreen shrub widely distributed in Europe (Brickell and Mathew 1976). Typically it grows in the undergrowth of shady mountain forests. In the Iberian Peninsula, the species is frequent in the northern Cantabrian and Pyrenean Mountains, characterized by Atlantic climate and de- ciduous and mixed forest, and also in the southern Mediterranean Betic Ranges, where it inhabits evergreen sclerophyllous and sub- sclerophyllous woodlands (Alonso et al. 2007). At this large ge- ographic scale, D. laureola exhibits among-population variation in reproductive system. Gynodioecy is the most common sexual system and prevails in the northwestern and southeastern Iberian populations but there are at least two disjunct, distant areas in the northeast and southwest of the Iberian Peninsula in which purely hermaphroditic populations exist (Alonso et al. 2007).

We studied the phylogenetic relationship between individ- uals from four hermaphroditic and seven gynodioecious Iberian populations of D. laureola (see Fig. 1 for locations). Those pop- ulations were a subsample of a broader survey conducted to de- termine the geographic variation in the proportion of female and hermaphrodite plants per population (Alonso et al. 2007). All of them have more than 100 individuals. Gynodioecious popu- lations were located in NW, NE, and S Iberian regions and their frequency of females (11%–54%; Table 1) encompassed the range

Figur e 1. Daphne laureola L. distribution map according to Meusel et al. (1978). Locations of the two Slovenian populations are marked in the distribution map and the 11 Iberian populations sampled are shown in more detail, with the three a priori geographical regions we distinguished indicated. Black and white dots denote gynodioecious and hermaphroditic populations, respectively, population codes

as in Table 1.

of variation recorded at the broad geographic scale (see Alonso et al. 2007 for details). Three hermaphroditic populations were selected from the NE region where they are frequent, and one from the S region where they are not so common. In addition, we included two European populations outside the Iberian Penin-

sula, one hermaphroditic and one gynodioecious, to better char-

acterizing the phylogeographic relationships by broadening the geographic distances (Fig. 1). The two populations were located in two different Slovenian mountain ranges, distant about 70 km from each other, and the hermaphroditic population was smaller (22 individuals) than the gynodioecious population (>100 indi-


Ta b l e 1 . Location, reproductive system, female percentage, ribotypes (ITS) and haplotypes (psbA-trnH and ndhF sequences combined)

of the Daphne laureola populations studied. Regions and population codes as in Figure 1.


Population (code)

Latitude N/Longitude W

Reproductive system

% females



S Iberia:

Huerta Vieja (HV)






Cuevas Bermejas (CB)






La Maroma (ST)






Hoyos de D. Pedro (CA)






NW Iberia:

Las Cruces (GA)






Bosque Pelon˜ o (BP)






NE Iberia:

Aldatz (AL)






Selva Villanu´ a (SV)






S. Juan de la Pen˜ a (SJ)






Gresolet (GR)






Tagamanent (TG)







ca. Vrhnika (SL1)






ca. Kocevje (SL3)






Each population was represented in the phylogenetic analy- ses by two individuals of each sex (N = 42 individuals) to allow detecting possible polymorphisms linked to sex (e.g., McCauley and Olson 2003). The small sample size per population was cho- sen on the assumption of reduced levels of polymorphism in DNA sequences at the within-population level, as frequently found for plastid markers (e.g., Cornman and Arnold 2007).

Finally, leaf samples from two to six individuals of other six European Daphne species (D. blagayana, D. cneorum, D. gnidium, D. mezereum, D. oleoides, and D. rodriguezii) were also collected and similarly analyzed to allow the rooting of the phylogenetic trees because no molecular phylogeny of Daphne is currently available.


Total genomic DNA was isolated from freshly frozen bud tissue maintained at −80 C, using a DNeasy 96 Plant Kit (QIAGEN, Inc., Valencia, CA). We selected one nuclear and two cpDNA re- gions for sequencing: the ITS region of the 18S-5.8S-26S nuclear ribosomal cistron and the psbA-trnH intergenic spacer and the ndhF gene, respectively.

PCR-amplification was performed by using PCR-Beads kits (PuReTaq Ready-To-GoTM , Amersham Biosciences, UK) with approx. 25 ng of DNA template, and 0.2 mM of each primer in a final volume of 25 μL. The following primers were used for each region: P1A/P4 for the complete ITS region (ITS1,

5.8S gene, ITS2; White et al. 1990; Downie and Katz-Downie

1996), psbA/trnH for their intergenic spacer (Sang et al. 1997), and primers 1318 and 2110R for the 3i end of the ndhF plastid gene (Olmstead and Sweere 1994). The PCR mix underwent the following conditions on a 9700 thermal cycler (Perkin–Elmer, Norwalk, CT): 10 min denaturing at 95 C, 37 cycles of 30 s de- naturing at 95 C, 30 s annealing at 52 C, and 1 min extension at

72 C and a final extension step at 72 C for 7 min. The PCR prod-

ucts were then purified using spin filter columns (UltraCleanTM PCR Clean-upTM Kit, MoBio Laboratories, Inc., Carlsbad, CA) following the protocols provided by the manufacturer and directly two-way sequenced with an ABI Prism BigDye Terminator Cycle kit on a ABI Prism 3730 (Applied Biosystems, Norwalk, CT). The resulting electropherographs were manually proofread and the complementary strands combined into one sequence to iden- tify ambiguous positions assisted by Sequencher 4.8 (Gene Codes Corporation 2007).

Sequences and alignment

Forty-two ITS sequences of D. laureola and 18 of the other Daphne species analyzed were generated from forward and reverse sequences (GenBank accession numbers GQ167491– GQ167548, HQ268822–HQ268823). Forty-two sequences of the

psbA-trnH marker were obtained for D. laureola, and 16 of the other Daphne species (GenBank accession numbers GQ167433– GQ167490). Thirty-seven sequences of the ndhF marker were obtained for D. laureola, and 10 of the other Daphne species (Gen- Bank accession numbers GQ167388–GQ167432, HQ268820– HQ268821). For sequence analyses, multiple alignments were performed using ClustalX, with default parameters for gap open- ing and extension (Thompson et al. 1997). The ITS and ndhF regions analyzed did not show any indel variation within the D. laureola sequences that constitute the focal purpose of this study (see Appendices S1 and S2). Consequently, gaps were treated as missing data. The alignment of the psbA-trnH intergenic spacer was not so straightforward. First, at the interspecific level a 5 bp inversion occurred at position 36, that was coded as a sin- gle character change, with all ingroup taxa exhibiting the same character state for the inversion. Second, the initial alignment of our study sequences (see Appendix S3) suggested that two in- sertion/deletion events occurred within the D. laureola sequences analyzed that would be ignored if treated as missing data. The first one involved an A rich region with 9, 10, or 11 repeats, and the second one involved a TA motif with 7, 10, or 13 repeats. The complex architecture of this plastid marker that can affect au- tomatic alignment (Storchova and Olson 2007; Morrison 2009), and the absence of sequences of other Thymelaeaceae phyloge- netically close to Daphne that could improve the phylogenetic interpretation of these events precluded us from coding indels and thus gaps were conservatively treated as missing data.
Haplotype detection

Statistical parsimony network implemented in the TCS program (Clement et al. 2000) was used to detect haplotypes, estimate the mutational differences among them justified by the parsimony criterion, and establish the resulting network that would allow incorporating the frequently nonbifurcating genealogical infor- mation associated with population-level divergences. Confidence level was set at 95%, and gaps were treated as missing data.

Phylogenetic analysis

Five ITS sequences of the sister genus Thymelaea (Galicia- Herbada 2006) retrieved from GenBank (AJ549483 T. vil- losa, AJ549489 T. argentata, AJ549468 T. dioica, AJ549470 T. granatensis, and AJ549442 T. passerina), were included into the analyses to explore the phylogenetic relationships of Daphne with respect to another Thymelaeaceae and improve the rooting of our focal sequences. Phylogenetic relationships among taxa were es- timated using maximum parsimony analysis (unordered charac- ters, equal weights; referred as MP hereafter) as implemented in PAUP 4.0b10 (Swofford 2002). Heuristic searches with random trees used as a starting point and 10 random addition sequences of taxa were performed with the TBR branch-swapping algorithm

and MulTrees options in effect. Bootstrap support (referred as BS

hereafter) values were calculated on 1000 replicates.

Bayesian inference phylogenetic analyses were also con- ducted using MrBayes version 3.1.2 (Huelsenbeck and Ronquist

2001). Analyses were run for 100,000 generations with four MCMC chains and two independent runs with trees sampled ev- ery 100th generation, and the first 80–90 sampled trees discarded by burn-in. Modeltest version 3.7 (Posada and Crandall 1998) was used to determine the DNA substitution model that best fitted the data among those available in MrBayes. The model chosen for our data using hierarchical likelihood ratio tests was HKY + G, which assumes a time-reversible process, a nonuniform distribution of nucleotides and different rates for transitions and transversions, and accounts for among-site rate variation using the gamma dis- tribution. Thus, we set Nst = 6 and rates = gamma options in MrBayes analyses.

We performed maximum likelihood ancestral state recon- structions of the population reproductive system on the MP tree based on the three sequenced regions analyzed simultaneously, that was topologically similar but with a better resolution than those obtained from cpDNA and rDNA markers analyzed inde- pendently. As reproductive system is a population-level feature, we reduced our sample to just a single individual per popula- tion. This procedure should not affect phylogenetic signal in any important way, because in most cases all the samples from the same population were invariant in sequence and exhibited identi- cal ribotypes and haplotypes (Table 1). In the only three popula- tions where polymorphism occurred at some marker, variability involved a single individual out of the four sampled, and we rep- resented each population using the commonest variant. We used Mesquite 2 (Maddison and Maddison 2007) and coded population sexual system as a binary character (hermaphroditism vs. gynodi- oecy). The rate of a character’s evolution was estimated under the simple Markov k-state one-parameter stochastic model, assuming that transition between hermaphroditism and gynodioecy had the same probability in both directions.


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