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 plants 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 Africa, 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 plant family Hyacinthaceae includes approximately 1,000 bulbous monocot species distributed throughout Africa, Europe, and southwest Asia, with one genus (Oziroe Raf.) in South America. 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 South Africa (Venter, 2009). The Ledebouriinae consist of three genera: Drimiopsis Lindl. & Paxton, Resnova Van der Merwe, and Ledebouria 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 Arabian Peninsula, Madagascar, Socotra, and India. 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 Scilla 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, inflorescence 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 clade 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 seed 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 taxonomy 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 bulb structure using microCT scans, floral volatile compounds, etc.).