The evolutionary history of the Ledebouriinae (Hyacinthaceae)

RESEARCH PROPOSAL

Principal investigator: Mr. Cody C. Howard, Florida Museum of Natural History, University of Florida

Abstract The Hyacinthaceae are a family of bulbous monocotyledonous primarily distributed throughout arid to semi-arid climates in the Old World. Within this group, members of the subtribe Ledebouriinae have been poorly represented in phylogenetic studies on the family as a whole. This has resulted in poor resolution within the Ledebouriinae, further producing questionable results pertaining to their evolutionary history. The use of next-generation sequencing, targeting rapidly evolving DNA markers will help to uncover the relationships within this subtribe, and allow for deeper investigations into this group’s evolutionary past. Continued research on the Ledebouriinae will also improve our understanding of its diversity, which will further enhance our ability to investigate topics related to genome evolution, polyploidy, and hybridization. Currently, the PI has obtained representatives of this subtribe from South , Namibia (past personal fieldwork), Mozambique, Uganda, Socotra, and Sri Lanka. Additional samples from both living and herbarium collections will be sought from herbaria such as Kew, the Missouri Botanical Garden, and the French National Museum of Natural History. Tanzania represents an important area that is not represented in current studies on the Ledebouriinae, and living collections from this region will improve our ability to resolve the relationships within the Ledebouriinae and will help us to improve our understanding of its historical dispersal across and out of Africa.

Introduction The family Hyacinthaceae includes approximately 1,000 bulbous monocot distributed throughout Africa, , and southwest Asia, with one ( Raf.) in . This group contains a high level of diversity in southern Africa as well as the Mediterranean region of Europe and northern Africa, with taxa predominately located in arid to semi-arid environments. Four distinct subfamilies exist within the Hyacinthaceae: Oziroeoideae, Ornithogaloideae, Urgineoideae, and Hyacinthoideae. Taxonomic uncertainty still remains an issue in many of these groups, and new species are still being discovered and described from across their distributions (Martínez-Azorín, et al., 2010, 2011, 2012a, 2012b, 2013a, 2013b, 2013c, 2013d, 2014a, 2014b, 2015a, 2015b, 2015c, 2015d; Pinter et al., 2012, 2015; Hankey, 2014; Cumming, 2015; plus many more species that reside outside of Africa). The subtribe Ledebouriinae (located within the subfamily Hyacinthoideae) have been poorly represented in phylogenetic studies pertaining to the Hyacinthaceae, despite the fact they are commonly encountered throughout their distribution in sub-Saharan Africa with the center of diversity currently located in the Limpopo, Mpumalanga, and KwaZulu-Natal regions of (Venter, 2009). The Ledebouriinae consist of three genera: Lindl. & Paxton, Van der Merwe, and Roth, with Ledebouria being of most interest. Drimiopsis and Resnova are both African endemics and commonly occur sympatrically with Ledebouria, but Ledebouria can also be found in the , Madagascar, Socotra, and . This interesting distribution may be due to a fascinating evolutionary history that still remains to be told.

Literature Review The type species of Ledebouria (L. hyacinthina Roth) was described from India (Roth, 1821). It was later transferred to L. by Baker (1870). Ledebouria then remained a subgenus of Scilla until 100 years later when Jessop (1970) reinstated it as its own genus for South Africa based on characters of the leaves, and flowers. Due to the widespread distribution of Ledebouria and variations in leaf spotting and growth habit, many name changes throughout the taxonomic history of this genus has occurred, leading to nomenclatural and taxonomic confusion. Previous phylogenetic studies of the Ledebouriinae have failed to provide phylogenetic resolution within this and have included only a small fraction of this group’s diversity, resulting in further taxonomic disagreement among researchers (e.g., Manning, 2004; Lebatha, 2006). The work of Stedje (1998) used a combination of morphological and plastid DNA analyses and confirmed that Jessop’s splitting of Scilla and Ledebouria was appropriate but did not attempt to resolve relationships within the Ledebouriinae since it was not the focus of the study. In 2003, Pfosser et al. investigated the phylogenetic relationships of the genera within the tribe Massonieae, in which the subtribe Ledebouriinae is found, and was unable to resolve the relationships within the Ledebouriinae based on plastid DNA and morphology. Later, the works of Manning (2004) and Lebatha (2006) specifically discussed the phylogenetic relationships that exist between Ledebouria, Drimiopsis and Resnova. Manning lumped Drimiopsis and Resnova into Ledebouria due to a polytomy along the backbone of this group. Like the other studies before his, he used only plastid DNA markers. Lebatha, using a combination of morphological and plastid DNA datasets (similar to Stedje (1998)), recovered competing phylogenies between the morphological and molecular datasets. The molecular datasets resulted in a paraphyletic Ledebouria, which agreed with Manning’s lumping of the group into one genus. In contrast, the morphological data produced a phylogeny that supported the separation of the three genera. Therefore, Lebatha suggested that the three genera be kept separate based on competing hypotheses and different data sets. Lebatha states that, “A revised of Ledebouria, Resnova and Drimiopsis is premature”, suggesting that more work is needed. As stated above, previous phylogenetic studies have failed to provide resolution within the Ledebouriinae and have included only a small fraction of this group’s diversity. Preliminary analyses conducted at the University of Florida, which included greater taxon sampling and using the plastid marker trnL-F (the same marker used in the studies mentioned above) still fail to provide resolution within this clade. This is potentially due to putative rapid speciation events leading to incomplete lineage sorting (ILS). Additionally, hybridization may be contributing to weak support and low resolution within this clade as sympatric populations are commonly encountered across the distribution. However, both cases have yet to be investigated in this group. Therefore, multiple, rapidly evolving markers are likely necessary to resolve relationships confidently within the group, in addition to greater taxon sampling from across its distribution. Consequently, since we are unable to resolve fully the relationships within this group and lack samples from across its distribution, investigations into the historical biogeography of this group are difficult to execute confidently. Past studies have produced questionable results with conflicting age estimates for the Ledebouriinae (25MA (Buerki et al., 2012) vs 6.3MA (Ali et al., 2012)). Ali (2012), using a dataset provided from Wetschnig (2007), suggested multiple long- distance dispersal events of Ledebouria from southern Africa; however, a large sampling gap from eastern, western, and northern Africa limits the support of this hypothesis. The authors even state that including samples from eastern Africa may provide a better picture of the historical dispersal of this group (i.e., Hyacinthaceae). Additionally, Pfosser (2012) shows a close relationship between an Indian and Madagascan Ledebouria species but a lack of samples from eastern Africa again lends low support to this hypothesis. Ledebouria is the only taxon in the Hyacinthaceae with species found in Africa, Madagascar, Socotra, the Arabian Peninsula, and India. Such a widespread distribution might be the product of a complex history and evolutionary processes that led to its current pattern. Therefore, by including samples from Tanzania we will be able to form a better hypothesis of the evolutionary history of the Ledebouriinae as a whole, and then begin to look more deeply into the successful dispersal of Ledebouria out of Africa.

Research Problem As stated above, samples from across the distribution of the Ledebouriinae are lacking, therefore limiting our ability to resolve fully the relationships within this subtribe of bulbous monocots and investigate its evolutionary past. I hope to collect species of this group, as well as other species within the Hyacinthaceae, in order to produce a more robust phylogeny to improve our evolutionary understanding of both groups.

Objectives 1) To collect living and herbarium samples of Hyacinthaceae from northern Tanzania. 2) To collect herbarium samples of associated species in other families in order to build ecological associations. 3) To build herbarium collections of Hyacinthaceae for deposition in Tanzanian and US herbaria. 4) To increase awareness of Hyacinthaceae research by publishing in high impact journals. 5) To disseminate products of this research to scientists and the general public in the form of journal articles, public speaking engagements, and museum displays.

Methodologies Note: exportation of living specimens are necessary in order to be able to fully characterize various morphological characters (e.g., internal structure using microCT scans, floral volatile compounds, etc.). Past experience in Namibia has shown that finding populations in flower and/or is rare, therefore limiting our ability to make complete and useful herbarium vouchers. Living collections can be exported to the US, and grown under greenhouse conditions until flowering. Upon completion of characterization, samples will be vouchered and sent to the targeted herbaria mentioned under Research Beneficiaries. I will assemble a comprehensive molecular dataset aimed at reconstructing the evolutionary history of the Ledebouriinae. To produce the most robust and accurate phylogeny, for each species I will include accessions from multiple populations and sequence numerous chloroplast and nuclear loci using next-generation sequencing techniques (Illumina). To this end, I am designing probes (through the company MYcroarray: www.mycroarray.com) for over 200 single-copy nuclear genes and most of the chloroplast genome that will allow me to enrich these genomic regions prior to sequencing. These probes will be designed from two available transcriptomes within the Hyacinthaceae from the 1KP Project (www.onekp.com), and seven genome sequence skims (low-coverage reads) of the Hyacinthaceae. After assembling the loci using a combination of de novo and reference-based methods, I will conduct phylogenetic analyses using a variety of approaches: gene trees for the concatenated plastid data; gene trees for the concatenated nuclear data; and species trees from all of the nuclear genes (biparental inheritance) treated as separate loci and plastid genes (uniparental inheritance) as a single locus. These approaches will allow for a more complete picture of evolution and will help to highlight any putative instance of hybridization. Datasets will be analyzed using Maximum Likelihood (RAxML (Stamatakis, 2014) and Bayesian coalescent (BEAST (Drummond et al, 2012)) approaches. Dating analyses will also be conducted using BEAST. For the morphological analysis I will select and score numerous characters across all species and will base my observations on both living and herbarium material. Characters will include phenotypes associated with adaptation to dry environments (i.e., leaf stature, bulb morphology, etc). Morphological data sets will then be analyzed independently and in combination with molecular data to generate phylogenetic and evolutionary inferences. From these analyses I will be able to assess which traits are likely linked to bursts of diversification within the group. Morphological character evolution will be investigated using ancestral state reconstruction methods as implemented in Mesquite (Maddison & Maddison, 2008) and the R package ape (Paradis et al, 2004). An in-depth look at evolution within the Ledebouriinae is also needed. Within the Ledebouriinae, chromosome counts range from n = 5 – 33 and this suggests multiple incidences of polyploidy and/or hybridization. Using the software chromEvol 2.0 (Glick & Mayrose, 2014) I can assess the diverse and remarkable evolution of ploidal levels in this group. I will obtain chromosome counts from root tip squashes for the living specimens collected. Samples collected from herbarium vouchers will have genome estimates measured using flow cytometry. By using a comparative approach of likelihood and Bayesian models implemented in BioGeoBears (Matzke, 2013), BAYAREA (Landis et al., 2013), and RASP (Yu et al., 2015), I will estimate ancestral ranges to investigate potential dispersal routes of the Ledebouriinae within Africa, and for Ledebouria, out of Africa. From these analyses I can trace the evolution and radiation of the group looking at the role of both dispersal and vicariance. Combining this knowledge with historical, geological, and climatic changes will elucidate the adaptive nature of these primarily arid-adapted taxa.

Research Beneficiaries Scientists and students will benefit from the improved collections of Hyacinthaceae that will be housed in the National Herbarium of Tanzania (NHT), the Florida Museum of Natural History (FLAS), the Huntington Botanical Gardens (HNT), and the Missouri Botanical Garden (MO). DNA voucher specimens reported in GenBank will be linked to herbarium specimens housed at these institutions, further increasing their value. Improved living collections will allow for ex situ conservation of potentially important species and allow for deeper genomic investigations (i.e., whole genome sequencing) by the PI. In addition to training undergraduate students in horticultural practices, as well as molecular and morphological systematic techniques, my research will generate a number of deliverables that will include a formal taxonomic treatment with keys to species with illustrations, digital and printed distribution maps, a research website, and journal articles (both scientific and non-scientific). Furthermore, by reaching over 200,000 visitors per year the Florida Museum of Natural History (FLMNH) provides an excellent venue to educate the public by allowing me to generate an exhibit highlighting the evolution of plants into arid and semi-arid habitats. Additionally, my affiliation with the Huntington Botanical Gardens (HNT) will allow me to present my research through their publication Huntington Frontiers, which reaches 41,000 members and visitors per issue. Overall, my intimate affiliation with two museums will heighten the public impact of my research. I will continue to pursue further outreach opportunities both locally and internationally, which will bring attention to FLMNH, HNT, and the ongoing research at these institutions, as well as the international collaborators that helped with this research.

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