A phylogeny of the Rutaceae and a biogeographic study of its subfamily Aurantioideae Thomas Schwartz Supervisor Bernard Pfeil Degree project for Master of Science In Systematics and Biodiversity, Biology 60 hec Department of Plant and Environmental Science University of Gothenburg Abstract The Rutaceae classification is complex and has undergone several changes. In addition to morphological studies, phylogenetic inference using molecular data has also led to classification changes. Thus far only chloroplast data and ITS have been used, sometimes combined with morphology to infer the phylogeny. This study adds information from a low copy nuclear gene to test the existing phylogenetic hypothesis using a species tree framework. A biogeographic study was also performed on the Aurantioideae subfamily. A pilot study looked at the choice of genes, followed by testing and evaluation of several methods for extraction of Rutaceae DNA. Thereafter, a new method for efficient separation of alleles and paralogues was examined. The sequences obtained were analysed for recombination, positive selection and hybridisation. Trees for three loci (chloroplast, nuclear HYB and MDH) were made using MrBayes and BEAST, and a species tree was constructed with *BEAST. The *BEAST species tree is used as a template for a biogeographic study with the Lagrange geographic range likelihood analysis. A Bayesian biogeographic study is also performed using a Bayesian discrete biogeographical mode (an addition to BEAST). The results are then compared with previous studies, corroborating some and rejecting others. Sammanfattning Rutace-familjens struktur är komplex och föränderlig. Förutom morfologiska studier har även fylogenetiska studier använts för att få ordning i den. Hittills har man endast tittat på kloroplastgener och ribosom-DNA, i enstaka fall i kombination med morfologiska karaktärer. Den här studien tillför nukleär-DNA från lågkopiegener samt en biogeografisk studie av dess underfamilj, Aurantioideae. En förstudie undersöker hur användbara generna som använts är. Detta följs av en undersökning av flera metoder för extraktion av DNA. Därefter testas en ny metod för att skilja på alleler och paraloga gener. De gensekvenser som tas fram analyseras för rekombination, riktad utveckling samt hybridisering. Fylogenetiska träd för de olika generna konstrueras med MrBayes och BEAST. *BEAST används för att göra ett gemensamt artträd. Detta artträdet blir sedan stommen i en studie där programmet Lagrange beräknar sannolikheten för hur den geografiska utbredningen har utvecklats. En bayesisk biogeografisk undersökning utförs också med BEAST. Resultaten jämförs till sist med tidigare forskning, där en del studier blir styrkta medan andra blir avvisade. Background Rutaceae is a varied and widely spread family of mainly tropical trees and shrubs (Scott et al., 2000). At least a few of the about 170 genera can be found on all continents except Antarctica. It is, however, not represented north of the Alps or in Canada and only poorly represented in Europe, central Asia and outside tropical parts of North America. The most well recognised subfamily, the Aurantioideae, includes all the citrus species (Mabberley, 1998). Other genera of economic interest are Pilocarpus, Boronia, Choisya, Poncirus and Skimmia (Chase et al., 1999). As can be seen in the table below, the phylogenetic history of Rutaceae is varied and uncertain. While it is recognised that the family, here broadly delimited (i.e., including Cneoraceae and Ptaeroxylaceae), is monophyletic (Chase et al., 1999, Gadek et al., 1996) , the internal classifications are contested (Scott et al., 2000). The first systematic treatment of Rutaceae, was made by (Engler et al., 1887) who divided the family into 6 subfamilies: Aurantioideae, Dictyolomoideae, Flindersioideae, Rutoideae, Spathelioideae, and Toddalioideae. These are further subdivided into a total of 25 tribes. Engler based his classification mainly upon flower and fruit morphology (Chase et al., 1999). Out of these subfamilies, the Aurantioideae is strongly supported as a monophyletic group (Samuel et al., 2001, Bayer et al., 2009). Engler remains the main authority on Rutaceae and the starting point of phylogenetic work on the family (Scott et al., 2000, Groppo et al., 2008) despite the new work. Subfamilies other than the Aurantioideae have been rearranged, generally by merging to form larger subfamilies as explained below. Different classifications have been suggested since the work of Engler, and many of the papers cited here have suggested changes. One classification which takes these suggestions in account is found at the Germplasm Resources Information Network (GRIN). A list of papers whose conclusions are accepted can be found at the the website (GRIN). Some new findings that have not been incorporated include parts of the tribe level conclusions in the (Morton, 2009) paper. I will use both the GRIN and the Engler classifications as a base for this study with some additional support from Swingle (Swingle and Reece, 1967) for the Aurantioid subfamily. The following table [Table 1] shows the classifications mentioned above. Previous studies have been mainly based on plastid genes (broadly defined), including rbcL, rps16, trnL-trnF, atpB-rbcL (Gadek et al., 1996, Chase et al., 1999, Scott et al., 2000, Groppo et al., 2008). Some, however, have also used nuclear genes, ITS1 and ITS2 in this case (Morton, 2009, Poon et al., 2007) . As Morton's (2009) analysis focused on Aurantioideae and Poon's (2007) on Rutoideae and Toddalioideae, a family-wide phylogeny using the nuclear genome is lacking. Rutaceae Rutaceae GRIN Engler GRIN Engler Swingle Toddalioideae- Aurantioideae- Toddalioideae Toddalieae Aurantioideae Aurantieae Toddaliinae Citreae Citreae Acronychia Acronychia Balsamocitrinae Citrinae Balsamocitrinae Halfordia Halfordia Aeglopsis Aeglopsis Skimmia Skimmia Afraegle Afraegle Toddalia Toddalia Balsamocitrus Balsamocitrus Phellodendron Phellodendron Aegle Aegle Aegle Amyridinae Citreae Citrinae Amyris Amyris Citrus Citrus Citrus Pteleinae Feronia Feronia Feronia Ptelea Ptelea Feroniella Feroniella Feroniella Brombya Limoniinae Oriciinae Atalantia Atalantia Atalantia Rutoideae- Xanthoxyleae Citropsis Citropsis Citropsis Evodiinae Naringi Naringi Herperethusa Bouchardatia Bouchardatia Paramignya Paramignya Paramignya Evodia Evodia Pleiospermium Pleiospermium Pleiospermium Fagara Fagara Severinia Severinia Severinia Geijera Geijera Triphasia Triphasia Triphasia Melicope Melicope Triphasiinae Orixa Orixa Monanthocitrus Monanthocitrus Sarcomelicope Sarcomelicope Wenzelia Wenzelia Zanthoxylum Zanthoxylum Burkillanthus Burkillanthus Boninia Swinglea Swinglea Choisyinae Microcitrus Microcitrus Choisya Choisya Poncirus Poncirus Dutaillyea Dutaillyea Clymenia Clymenia Medicosma Medicosma Oxanthera Oxanthera Decatropidinae Pamburus Pamburus Megastigma Clauseneae Lunasiinae Merrilliinae Merrilliinae Lunasia Lunasia Merrillia Merrillia Pitaviinae Clauseniae Rutoideae-Ruteae Murraya Murraya Murraya Dinosperma Clauseneae Dictamninae Bergera Bergera Dictamnus Dictamnus L. Clausena Clausena Clausena Euodia Glycosmis Glycosmis Glycosmis Pitaviaster Micromelinae Tetradium Micromelum Micromelum Micromelum Flindersioideae- Rutoideae-Boronieae Flindersioideae Flindersieae Boroniinae Flindersia Flindersia Myrtopsis Myrtopsis Rutoideae-Ruteae Boronieae Rutoideae Rutinae Acradenia Acradenia Ruta Ruta Boronia Boronia Dictyolomatoideae Zieria Zieria Spathelioideae -Dictyolomateae Cneorum Eriostemoninae (CNEORACEAE) Asterolasia Asterolasia Dictyoloma Dictyoloma Harrisonia Eriostemon Eriostemon (SIMAROUBACEAE) Neochamaelea Phebalium Phebalium (CNEORACEAE) Spathelioideae- Philotheca Philotheca Spathelieae Nematolepidinae Nematolepis Nematolepis Correinae Correa Correa Rutoideae-Cusparieae Diplolaeninae Diplolaena Diplolaena Cusparieae Cuspariinae Erythrochiton Erythrochiton Ravenia Ravenia Pilocarpinae Pilocarpus Pilocarpus Nycticalanthus Rutoideae-Diosmeae Diosmeae Diosminae Agathosma Agathosma Empleurinae Empleurum Empleurum Calodendrinae Calodendrum Calodendrum Sheilanthera lacks subfamily and tribe Boninia Table 1: The classifications of Rutaceae made by Engler 1986, GRIN 2010, and Swingle's subfamily Aurantioideae classification. The table includes those genera that have been part of this thesis in either analysis or extraction. Family, subfamily and tribe are underlined. The tribes are in italic. Single gene phylogenies can be misleading (not track the species phylogeny) because of selection e.g., (Stefanović et al., 2009), mistaken orthology (e.g., (Straub et al., 2006)), lineage sorting (e.g., (Avise et al., 1983)), hybridisation (e.g., (Cronn and Wendel, 2004)) and recombination (e.g., (Sanderson and Doyle, 1992)). Therefore, generating sequence from more than one gene, and especially from more than one linkage group, is the basic information required to infer species trees accurately (Edwards et al., 2007). Low-copy nuclear genes (those perhaps least subject to concerted evolution) may provide the best source of additional genes not linked to the chloroplast genome to complement existing phylogenetic evidence in Rutaceae. The aim of this paper, then, is to achieve a better estimate of the phylogeny for Rutaceae, built on the low-copy nuclear genes HYB (beta-carotene hydroxylase) and MDH (malate dehydrogenase). Species of interest The main objective of this paper is to verify the classifications made for the Rutaceae family. This mainly points to the subfamilies