University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications in the Biological Sciences Papers in the Biological Sciences 4-2008 A 15-Myr-Old Genetic Bottleneck Timothy Paape University of California, San Diego Boris Igic University of Illinois at Chicago Stacey DeWitt Smith University of Nebraska - Lincoln, [email protected] Richard Olmstead University of Washington Lynn Bohs University of Utah See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/bioscifacpub Part of the Life Sciences Commons Paape, Timothy; Igic, Boris; Smith, Stacey DeWitt; Olmstead, Richard; Bohs, Lynn; and Kohn, Joshua R., "A 15-Myr-Old Genetic Bottleneck" (2008). Faculty Publications in the Biological Sciences. 116. https://digitalcommons.unl.edu/bioscifacpub/116 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications in the Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Timothy Paape, Boris Igic, Stacey DeWitt Smith, Richard Olmstead, Lynn Bohs, and Joshua R. Kohn This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ bioscifacpub/116 Published in Molecular Biology and Evolution 25:4 (April 2008), pp. 655-663. Copyright © 2008 Timothy Paape, Boris Igic, Stacey D. Smith, Richard Olmstead, Lynn Bohs, and Joshua R. Kohn. Published for Society for Molecular Biology and Evolution by Oxford University Press. Used by per- mission. DOI: 10.1093/molbev/msn016. Accepted December 28, 2007; published April 2008; posted to the University of Nebraska-Lincoln Digital Commons December 2010. A 15-Myr-Old Genetic Bottleneck Timothy Paape,1 Boris Igic,2 Stacey D. Smith,3 Richard Olmstead,4 Lynn Bohs,5 and Joshua R. Kohn1 1. Section of Ecology, Behavior and Evolution, Department of Biological Sciences, University of California, San Diego ([email protected]) 2. Department of Biological Sciences, University of Illinois at Chicago 3. Department of Biology, Duke University (2010: University of Nebraska-Lincoln, [email protected]) 4. Department of Biology and Burke Museum, University of Washington 5. Department of Biology, University of Utah Abstract Balancing selection preserves variation at the self-incompatibility locus (S-locus) of flowering plants for tens of millions of years, making it possi- ble to detect demographic events that occurred prior to the origin of extant species. In contrast to other Solanaceae examined, SI species in the sis- ter genera Physalis and Witherinigia share restricted variation at the S-locus. This restriction is indicative of an ancient bottleneck that occurred in a common ancestor. We sequenced 14 S-alleles from the subtribe Iochrominae, a group that is sister to the clade containing Physalis and Witherin- igia. At least 6 ancient S-allele lineages are represented among these alleles, demonstrating that the Iochrominae taxa do not share the restriction in S-locus diversity. Therefore, the bottleneck occurred after the divergence of the Iochrominae from the lineage leading to the most recent com- mon ancestor of Physalis and Witherinigia. Using cpDNA sequences, 3 fossil dates, and a Bayesian-relaxed molecular clock approach, the crown group of Solanaceae was estimated to be 51 Myr old and the restriction of variation at the S-locus occurred 14.0-18.4 Myr before present. These re- sults confirm the great age of polymorphism at the S-locus and the utility of loci under balancing selection for deep historical inference. Keywords: Balancing selection, Genetic bottleneck, S-locus, Self-incompatibility, Shared ancestral polymorphism, Solanaceae Introduction leles accumulating until a balance is reached between selec- tion favoring rarity and drift causing allele loss (Wright 1939; Theoretical population genetic studies of balancing selec- Lawrence 2000). Second, alleles are often very old because, tion predict that it will greatly increase the coalescence time if any allele drifts toward rarity, selection acts to increase its of allelic polymorphism relative to neutral variation (Taka- frequency (Ioerger et al. 1990; Clark 1993). In the Solanaceae, hata 1990; Vekemans and Slatkin 1994). This prediction has the S-locus gene responsible for self-pollen recognition and been confirmed by studies of self-recognition loci such as rejection in the female tissue is an RNase (S-RNase hereafter; the MHC loci of jawed vertebrates (Klein et al. 1993) and the McClure et al. 1989). The great age of polymorphism at the S- mating compatibility loci of both fungi (Muirhead et al. 2002) locus is exemplified by the fact that S-RNase alleles from the and plants (Ioerger et al. 1990; Richman and Kohn 2000; Cas- same diploid individual of Solanaceae often differ at more tric and Vekemans 2004). In all these systems, the time to co- than 50% of their amino acid sites. In addition, S-RNase al- alescence of allelic variation is far older than extant species. leles from species in different genera often cluster together in Loci under balancing selection can therefore provide evi- phylogenetic analyses, evidence of broadly shared ancestral dence of historical genetic and demographic events that far polymorphism (Ioerger et al. 1990; Richman and Kohn 2000; predate current species, a utility that has been termed ‘‘mo- Igic et al. 2004, 2006; Savage and Miller 2006). Much of the S- lecular paleopopulation biology’’ (Takahata and Clark 1993). locus polymorphism found in SI Solanaceae was present in In many flowering plants, self-incompatibility (SI) sys- their common ancestor, which must also have been SI (Igic et tems allow hermaphroditic individuals to recognize and re- al. 2004, 2006). ject their own pollen in favor of pollen from other individu- A striking contrast exists between the sequence diversity als, thus avoiding the deleterious effects of self-fertilization of S-alleles from species of the closely allied genera Physalis (de Nettancourt 1977). In single-locus gametophytic SI, as and Witherinigia, and nearly all other Solanaceae, whose S-al- found in the Solanaceae (nightshade family) studied here, leles have been sampled (species of Brugmansia, Lycium, Ni- a match between the S-allele carried by the haploid pollen cotiana, Petunia, and Solanum). Although the numbers of S-al- grain and either of the S-alleles in the diploid style triggers leles present in Physalis and Witherinigia species are similar pollen tube rejection, preventing self-fertilization and also to those found in other Solanaceae (Lawrence 2000; Stone cross-fertilization if the cross-pollen grain carries either al- and Pierce 2005; Savage and Miller 2006; Igic et al. 2007), all lele found in the female parent. In such systems, rare alleles 93 S-RNases sequenced from 3 Physalis (Richman et al. 1996a; have a selective advantage because they are compatible with Richman and Kohn 1999; Lu 2001) and 2 Witherinigia (Rich- more mates (Wright 1939). Selection favoring rare alleles is man and Kohn 2000; Stone and Pierce 2005) species cluster quite strong, even with large numbers of alleles segregating within only 3 S-allele lineages that predate the divergence of in populations. For instance, a new pollen S-allele entering a Physalis and Witherinigia. For other Solanaceae, even small population that already contains 20 alleles has an 11.1% male samples of alleles usually represent many more ancient lin- mating advantage (Clark 1993). eages (reviewed in Richman and Kohn 2000; Castric and Strong negative frequency-dependent selection is respon- Vekemans 2004; see also Savage and Miller 2006; Igic et al. sible for the 2 outstanding features of S-locus polymorphism. 2007). This finding has been interpreted as evidence of an First, dozens of alleles occur in natural populations with al- ancient bottleneck that restricted variation at the S-locus in 655 656 PAAPE ET AL. IN MOLECULAR BIOLOGY AND EVOLUTION 25 (2008) some common ancestor of the genera Physalis and Witherin- of 3’-RACE as in Igic et al. (2007). The forward degenerate igia. No such restriction is evident at the S-locus of any other primer PR1 (5’-GAATTCAYGGNYTNTGGCCNGA-3’) am- sampled SI Solanaceae (Richman et al. 1996b; Richman 2000; plifies from the 5’ end of the conserved region C2 (Ioerger Richman and Kohn 2000; Igic et al. 2004; Stone and Pierce et al. 1991) to the 3’ end of the coding region of the S-RNase 2005; Igic et al. 2006) except for African species of the genus cDNA. Products obtained via PCR were cloned using the Lycium (Miller et al. 2008), whose S-locus shows evidence of TOPO TA Cloning Kit (Invitrogen Corp., Carlsbad, CA) to a bottleneck associated with colonization of the Old World separate alleles at the obligately heterozygous S-locus. Am- from America. After the restriction of S-allele diversity in plified cloned PCR products were screened by restriction di- some common ancestor of Physalis and Witherinigia, the re- gests (10 clones per individual, on average) and sent for au- maining S-allele lineages diversified leaving the observed tomated sequencing by Eton Bioscience Inc. (San Diego, CA). pattern of large numbers of S-alleles representing only a re- stricted number of ancient S-allele lineages. Genealogy of S-alleles from Solanaceae In this paper, we date the historical restriction of S-locus variation common to Physalis and Witherinigia. First, we ex- For phylogenetic analysis of S-RNase sequences from the amine S-locus diversity in the South American monophy- Iochrominae, additional S-alleles were obtained from Gen- letic subtribe Iochrominae, which is found to comprise the Bank for the following species (number of alleles): Lycium an- sister group of the lineage containing Physalis and Witherin- dersonii (10), Nicotiana alata (6), Petunia integrifolia (6), Physalis igia by Olmstead et al. (forthcoming). We ask whether the Io- cinerascens (12), Solanum carolinense (9), and Witherinigia sola- chrominae share the reduced set of S-allele lineages found in nacea (15) (see Supplementary Material online for GenBank Physalis and Witherinigia.