A phylogeny of the Rutaceae and a biogeographic study of its

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
GRIN
Rutaceae
Engler
GRIN
Engler
Swingle
ToddalioideaeToddalieae
Aurantioideae
Toddaliinae
Citreae
Acronychia
Acronychia
Balsamocitrinae
Halfordia
Halfordia
Aeglopsis
Aeglopsis
Skimmia
Skimmia
Afraegle
Afraegle
Toddalia
Toddalia
Balsamocitrus
Balsamocitrus
Phellodendron
Phellodendron
Aegle
Amyridinae
Citreae
Amyris
Citrus
Citrus
Citrus
Pteleinae
Feronia
Feronia
Feronia
Ptelea
Feroniella
Feroniella
Feroniella
Toddalioideae
Amyris
Ptelea
AurantioideaeAurantieae
Citreae
Citrinae
Aegle
Balsamocitrinae
Aegle
Citrinae
Limoniinae
Brombya
Oriciinae
Atalantia
Atalantia
Atalantia
RutoideaeXanthoxyleae
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
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
Triphasiinae
Clauseneae
Megastigma
Lunasia
Lunasiinae
Merrilliinae
Merrilliinae
Lunasia
Merrillia
Merrillia
Pitaviinae
Rutoideae-Ruteae
Clauseniae
Murraya
Murraya
Clauseneae
Dinosperma
Dictamninae
Bergera
Bergera
Murraya
Dictamnus
Dictamnus L.
Euodia
Clausena
Clausena
Clausena
Glycosmis
Glycosmis
Glycosmis
Micromelinae
Pitaviaster
Tetradium
Myrtopsis
Micromelum
Micromelum
Rutoideae-Boronieae Flindersioideae
FlindersioideaeFlindersieae
Boroniinae
Flindersia
Flindersia
Rutoideae-Ruteae
Myrtopsis
Boronieae
Rutoideae
Rutinae
Ruta
Ruta
Zieria
Spathelioideae
Dictyolomatoideae
-Dictyolomateae
Eriostemoninae
Cneorum
(CNEORACEAE)
Asterolasia
Asterolasia
Dictyoloma
Eriostemon
Eriostemon
Harrisonia
(SIMAROUBACEAE)
Phebalium
Phebalium
Neochamaelea
(CNEORACEAE)
Philotheca
Philotheca
Acradenia
Acradenia
Boronia
Boronia
Zieria
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
Dictyoloma
SpathelioideaeSpathelieae
Micromelum
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 and tribes that are found in (Engler, Krause et al. 1887) and GRIN.
To do this successfully, several species and genera from a each subfamily will have to be obtained.
Each subfamily should be sufficiently represented to form a clade in a final tree, which would
require at least two individuals. Larger subfamilies would need a wider representation than small
subfamilies. Further, attempts were made to connect with previous work in the group by finding
samples of the same species used before, in an effort to test those earlier studies. Suggestions
gathered from these papers (Chase et al., 1999, Groppo et al., 2008, Poon et al., 2007, Pfeil and
Crisp, 2008, Morton et al., 2003, Morton, 2009, Salvo et al., 2008, Mole et al., 2004, Ling et al.,
2009, Samuel et al., 2001, Scott et al., 2000, Bayer et al., 2009) were influential in the gathering of
an ideal species sample list. Many of these papers have looked at samples from the Aurantioideae
subfamily but also the Toddaliinae and the Australian part of Boronieae. The Aurantioideae are also
good points of reference when working through the wider Rutaceae, considering that many genomic
resources, including primers for several low-copy nuclear genes, are available for the Aurantioid
genus Citrus. Other genera that have been extensively studied are Ruta, Skimmia and Zanthoxylum.
Other questions that may be answered by this study include Chase (1999) suggestion to look into
the relationship between Flindersia and Chloroxylon. There are also a group of genera that have
been moved out of Rutaceae. These includes Cedrelopsis and Ptaeroxylon, moved to
Ptaeroxylaceae; Cneorum and Neochamaelea, moved to Cneoraceae; and Harrisonia, moved to
Simaroubaceae. Some genera have also been placed outside of both subfamily and tribe in the
GRIN classification and are therefore phylogenetic orphans in search of a home clade. These are
Ivodea, Kodalyodendron, Megastigma, Tractocopevodia and Pseudoisma. Kodalyodendron is
endemic to Cuba and, since I have found no contemporary references to it, possibly extinct.
Geographical concerns
The question of whether to take account of geology / geography and climate and how to do it must
be answered in a study which wishes to discuss speciation and the historical spread of species. The
present continental settings relevant here were more or less formed between 40 and 20 Ma ago
(Irving, 1983). India had begun its collision with Eurasia at about 50 Ma and the archipelago
between Eurasia and Australia started forming around 40 Ma (Daly et al., 1991) and would reach its
conclusion when the Australian and Philippine plates met at 25 Ma (Baillie et al., 2004). Therefore
the fossil calibrated age of the Aurantioideae (Pfeil and Crisp, 2008) of around 20 Ma is
comfortably younger than any of the continental movements that might otherwise be related to the
spread of the Aurantioids. All of the Aurantioids live south or east of the Himalayas, therefore
suggesting that vicariance through mountain formation is not an issue. The rise and fall of islands
between Malaysia and Australia could possibly be related to the speciation process, even though
this would probably require the simultaneous disappearance of archipelagos, which does not seem
to have happened. All the considerations mentioned above gives leave to leave major geological
changes out of this study.
There is also the climate aspect to consider. Studies have shown that the Sahara desert has varied
between its present dry state and tropical xerophytic shrub-land (Pound et al.) or woodland
(PICKFORD et al., 2006) around 11 Ma. More recently there have been green periods in parts of
the desert during the archaeological time frame (Claussen and Gayler, 1997, Kuper and Kröpelin,
2006). What is known, then, is that the climate changes greatly, even during relatively short periods
of time. Such weather patterns would regularly open and close dispersal routes between Africa and
Asia, and quite likely similarly within Australia. However, the difficulty in simulating even the
present weather conditions accurately, let alone those from thousands of years back (de NobletDucoudré et al., 2000) means that trying to find the patterns and link them to phylogenetic work
surely would be a work worthy of Sisyphus.
Pilot Project
Methods
To examine the utility of the HYB and MDH genes for a Rutaceae phylogeny, a pilot test was
performed. Plants belonging to 13 different genera (Orixa sp., Phebalium squamulosum, Ptelea
trifoliata, Tetradium daniellii, Zanthoxylum bungeanum, “Skimmia sp.”, Ruta graveoleus,
Phellodendron japonicum, Correa decumbens, Dictamnus albus, Ailanthus altissima, Erythrochiton
brasiliensis and Murraya paniculta) grown in the Gothenburg Botanical Garden were collected and
dried in silica gel. They were extracted using the OMEGA Bio-Tek's EZNA Plant DNA MiniPrep
kit by the manufacturers instructions. The resulting DNA was then amplified through PCR using a
HotStartTaq DNA polymerase mix with the following concentrations: (per sample: Buffer *10;
2.5ul, MgCl2; 0,5ul, dNTP; 0,5ul, Primer 1; 1ul, Primer 2; 1ul, HotStart; 0,125ul, H2O; 19,4ul), and
then run in the PCR using the following scheme: (95C:5min, 94C:0.30min, 54C:0.30min,
72C:1min, 72C:5min, 4C=>infinity). The samples were amplified for HYB with the F120 and
R635 primers and for MDH with the F1 and R1 primers (Table 2). The samples were cleaned with a
QIAquick spin purification procedure and their DNA quality and concentration was assessed with a
Pharmacia Genequant II before being sent to Macrogen (Korea) for sequencing.
Nine (Orixa sp., P. trifoliata, T. daniellii, Z. bungeanum, “Skimmia sp.”, R. graveoleus, C.
decumbens, D. albus, and M. paniculta) of the locally extracted specimens resulted in successful
amplification of MDH and two (D. albus, M. paniculta) in amplification of HYB. Additional
amplifications were performed using previously extracted DNA mainly from Aurantioideae species
(Citrus gracilis, Citrus wintersiae Choisya ternata, Glycosmis maritiana, Glycosmis trichanthera,
Merrillia caloxylon, Atalantia monophylla, Clausena hamandiana, Paramignya lobata, Bergera
koenigii, Monanthocitrus cornuta, Murraya paniculta, Aegle marmelos, Afraegle paniculata,
Pamburus missionis, Balsamocitrus dawei, Aeglopsis chevalieri, Melicope elleryana, Philotheca
deserti). A total of 17 samples (A. marmelos, A. chevalieri, A. monophylla, B. dawei, B. koenigii, C.
ternata, C. gracilis, C. hamandiana, G. maritiana, M. caloxylon, M. elleryana, M. cornuta, M.
paniculta, P. missionis, P. lobata, and P. deserti) were amplified for the HYB gene. Sixteen of them
(excluding A. paniculata) were found to be of a sufficient quality to sequence and 14, excluding C.
ternata and G. maritiana, were successfully sequenced. The results from Bergera were of an
uncertain quality.
Alignments of MDH and HYB were made using Muscle (www.ebi.ac.uk/Tools/msa/muscle/), then
manually edited and corrected in BioEdit. MDH was represented by 24 samples in 14 genera,
whereas HYB was represented by the 13 specimens mentioned above, excluding Bergera. Two of
the MDH samples were designated as out-groups, whereas HYB had a Citrus mRNA sequence
added from GenBank (AY623047). Using PAUP* (Swofford, D. L. 2002.) the number of
parsimony-informative characters was determined with the command CStatus. Distances between
the sequences was calculated using the command DScores with default settings. The HYB data
were examined with two different alignments: the first included the full alignment, whereas the
second excluded areas containing lengthy off-setting of sections that were difficult to align, as well
as the last 200 bases that was also difficult to align. Every species examined had two sequences for
the HYB gene with unknown correct allele alignment. The alleles were compared within the clades
as discussed below.
Results and Discussion
The analyses showed that the percentage of parsimony informative bases were sufficient for both
genes. The MDH gene had 160 out of a total of 1163 (14%) parsimony informative (p.i.) characters
in this alignment. The two HYB alignments did even better with 287 p.i. characters out of a 1162
total characters (25%) for the HYB which included exon and stable intron, while the full HYB
alignment had 431 p.i. characters out of a total of 2126 characters (20%).
The distance matrix results were generally low for all alignments: MDH had an average of 6%, the
limited HYB an average of 9% and the full HYB alignment an average of 11%. Melicope, which
(together with Philotheca for the HYB alignments) was the only non-Aurantioid in this alignment,
stood out in all three alignments. It scored 11% to 14% points above the average for MDH, 5%
points above the average Aurantioid for the limited HYB alignment and between 3% and 6% points
above average for the full HYB. Melicope stood above average against Philotheca as well in the
limited HYB comparison, although not as markedly as compared to the other species.
The full HYB alignment had some further high scores that were lacking for the other alignments.
One notable difference was an increase in distance levels for Atalantia and Murraya, especially then
against Paramignya. Here one of two species of Atalantia proposed alleles had a 5% difference to
Paramignya compared with the other proposed allele. That allele also stood out in comparison with
the clade containing Aegle, Aeglopsis and Balsamocitrus. Murraya generally got higher distance
scores than Philotheca.
Generally, it appeared likely that both of these genes would provide suitable information for a
phylogenetic analysis of the family.
Main Project
METHODS
Species were initially sought after according to the criteria mentioned above. However, the main
criteria for species selection was that fresh material is better than dry material, which in practical
terms limited species selection to those species which are grown in botanical gardens. The
herbarium material gathered at the Missouri Botanical Gardens herbarium at TROPICOS® as well
as the silica gel collections at the National History Museum in Paris were investigated, however
requests were made too late to be used in this project. A list of acquired species can be found in
under Tables and Trees in an appendix.
Sampling and Processing
The first group of samples which were all gathered in the Gothenburg Botanical Garden, were
extracted using an (1) Omega bio-tek EZNA sp plant DNA Miniprep kit according to the
manufacturer's protocol (Ext23). The kit was thereafter used for extracting 12 specimens from the
Herbarium GB (Ext24).
CTAB I (2) To get a better result with fragile herbarium specimens, as well as with those fresh leaf
samples that had resisted kit extraction, a CTAB manual extraction was performed (Ext25). Seven
of the herbarium samples were then extracted using a modified CTAB protocol together with four
fresh leaf samples. The modifications included adding PVP-40, DIECA and Ascorbic acid to the
standard CTAB recipe (Doyle and Doyle, 1990). After drying but before re-suspending the samples
in TE, they were treated with proteinase K in a 1mM TRIS solution.
Searching for extraction processes
Having a poor result from the standard CTAB protocol mentioned above, a number of earlier
studies regarding DNA extraction chemistry were reviewed: (Kreader, 1996, Arif et al., 2010,
Alzate-Marin et al., 2009, Crouse, 1987) extraction protocol review articles (Drábková et al., 2002,
Ribeiro and Lovato, 2007) as well as other articles dealing with extractions from herbarium material
(Rogers and Bendich, 1985, Puchooa and Khoyratty, 2004). Ribeiro's article mentioned successful
extraction from herbarium material. It also mentioned successful extraction from Flindersia and
Melicope, two of the Rutaceae genera that I aimed to extract. The original article (Scott and
Playford, 1996) was found and the ingredients that were different from the original protocol were
located or bought. Preparing most of the buffers caused no problems. However, lacking a 5M NaCl
solution and failing to make one myself, the first (S&P) extraction (Ext26) used the same CTAB
solution as was used in the previous extraction and changed the RNAse purification step to a
proteinase K purification (adding 100 ul 1mM CaCl2 and 2.5 μl proteinase K, incubating for 15
minutes and deactivation at 95C for 10 minutes). It followed the protocol in all other respects
including grinding the samples in sand. The following extraction (Ext26b) tried to grind the samples
in liquid nitrogen. The resulting pellets had a much to gelatinous consistency, wherefore the
following extractions aimed at reducing this problem.
The two articles (Ribeiro, Scott and Playford) presented the protocol in different ways regarding
how specimens should be prepared. Therefore both liquid N2 and room temperature sand grinding
were tried and compared in the next step (Ext26c) which also used a 2M NaCl CTAB in which all
other ingredients were according to the recipe. Silica gel dried samples of four species growing in
the botanical garden were extracted. A different phenomenon was noted regarding the EtOH
precipitation, where washing with 1 volume of EtOH showed generous amounts of precipitate as
long as it was in a separate phase from the extraction supernatant but re-entered solution once the
tubes were mixed. These concerns together with generally weak results in 260/280 absorbance tests
and remaining concerns regarding gelatinous (see results) pellets led to further study of the theory
behind the process. Going back to the chemistry, further adjustments to the CTAB solution were
made. I identified the purpose each ingredient had on DNA extraction and compared it with the
listed ingredients of the kit I had done my first extraction with. Concluding from basic extraction
tutorials found on the internet (e.g. basic extraction demo's where adding NaCl to ground meat
produces extractable DNA) that a certain level of NaCl was necessary for successful DNA
extraction, I realised that the final NaCl concentration was diluted by 1/3 through adding 1ml CTAB
to 0.5 ml wash buffer.
The standard CTAB protocol uses 2M NaCl, which means that at least 3M NaCl is necessary for
successful extraction through the Scott and Playford protocol. I came to the conclusion that NaCl
was the key and made a new attempt at the CTAB solution. First making a 3M NaCl and then
adding NaCl until I approached 4M NaCl, although all of the salt in the latter did not enter solution.
Ext26d had the 4M NaCl CTAB solution and used N2 grinding. It also experimented on the effects
of using the initial extraction buffer twice and the CIA cleaning was performed twice as is common
in CTAB extractions. Everything went well until the drying step after the EtOH precipitation step.
Despite staying in the fume hood overnight, drops were visible on the inside of each tube. I
assumed that substances which should have been removed in the cleaning step but had remained
were the cause drying had been prevented and thus the EtOH step was repeated. I concluded that
both the NaCl and EtOH concentrations needed to be sufficiently high for successful extraction,
since the NaCl concentration was instrumental in producing pellets which were not encapsulated in
gelatinous substances and the EtOH concentration was required for a real precipitation to occur.
The Ext26e extraction tested whether precipitating in 2 or 3 volumes of EtOH would give the best
results. The leafs were ground in N2 and several of them (i.e. 3,4,5,6, which belonged to
Phellodendron samples) had a consistency approaching that of syrup in the extraction buffer. All
samples were to produce pellets given enough time in the centrifuge (15 minutes proved to short to
form pellets). After drying over night, the tubes had what looked like dried salt patches around the
tube mouths. (I have later realised that what I took to be salt may well have been DNA). They were
therefore washed again in EtOH.
The following extraction (Ext26f) changed direction on my extraction experiments, going back to
an initial 1 volume precipitation that was followed by a lengthy (23 min) centrifugation step. Two of
the samples (1,4) formed pellets and were washed in 75% EtOH whereas the remaining specimens
were centrifuged an additional five minutes. 500 ul liquid was then removed from the samples and
replaced with 96% EtOH before another 15 min of centrifugation commenced. This resulted in two
(3,5) more tubes having pellets, which were promptly washed. The remaining two tubes had the
liquid replacement step repeated. No pellet or DNA was ever recovered from these tubes.
Using liquid N2 for extraction purposes also proved to be disadvantageous. The Phellodendron
samples consistently produced an extraction buffer with the consistency of syrup, and extractions
where sand grinding had been used would still result in a thick extraction buffer if it was exposed to
freezing temperatures in the lab freezer. This probably reduced the extraction efficiency. This was
later confirmed by K.D. Scott through personal communication.
To determine whether the white cloud which forms in the 96% EtOH phase when it is added in the
precipitation step is DNA, Ext27 was performed. The precipitation step was performed in three
steps. First cold EtOH was carefully added, making sure that it did not mix with the supernatant
from the CIA cleaning step. The EtOH was thereafter removed to a new tube. A second portion of
EtOH was added. After a 30 minute incubation in the freezer, this EtOH phase was also removed to
a new tube. The two tubes with EtOH and the tube containing the remaining supernatant were then
pelleted.
The next extraction Ext28 tested whether more careful removal of the supernatant following the
CIA cleaning step would provide better results. Usually the samples had a white layer between the
CIA and the supernatant and a green slimy layer floating on top of the supernatant. Therefore two
supernatants were gathered from each sample. First the top 200 μl of the supernatant was removed
to the “b” tube, followed by 500 μl being removed to the “a” tube. Then the remaining supernatant
was added to the “b” tube. The tubes were initially precipitated in 2 volumes of cold 96% EtOH. All
samples except number 4 failed to provide pellets, instead producing a lower transparent phase near
the bottom of the tube. 800 μl of the upper phase was replaced with 70% EtOH in each of these
samples. They were then centrifuged again resulting in large pellets.
The final extraction (Ext29) was mainly a final try to add species to the list of obtained sequences.
It implemented the 2 supernatant transfers and adding and removing an EtOH phase before proper
precipitation. Unfortunately, I forgot to add the Ammonium acetate from the beginning of the
precipitation step, an oversight which caused a confused precipitation procedure. In the end, pellets
for both supernatant phases as well as for the EtOH was had been obtained.
Finally I came to the protocol that follows, which follows Ext28.
CTAB II (3) A CTAB protocol designed for tropical species (Scott and Playford, 1996) was further
modified and used as follows: approx. 30 – 50 mg of leaf tissue was ground in 2 ml extraction
buffer (50 mM Tris-HCl, pH 8.0; 5 mM EDTA; 0.35 M sorbitol; 0.1% bovine serum albumin [BSA]
and 10% polyethylene glycol, mol wt 6000) and approx. 0.2 g sea sand. This mix was centrifuged at
≥ XX,000 g for 5 min, the supernatant discarded and the pellets re-suspended in 400 µl of wash
buffer (50 mM Tris-HCl, pH 8; 25 mM EDTA and 0.35 M sorbitol) with 100 µl of 5% sarkosyl
solution added immediately thereafter. The tubes were then incubated at room temperature for 15
min.
1 ml of CTAB buffer (0.05 M CTAB; 1 M Tris-HCl, pH 8.0; 0.5 M EDTA; approx. 4 M NaCl) was
added and mixed by inversion. Scott and Playford's (1996) protocol lists 0.5 M CTAB at this step,
but this is in error (K.D. Scott pers. comm.). The solution was then incubated at 55C for ≥ 30
minutes.
Warming the combined solution for a few minutes makes the CTAB easier to mix with the previous
buffer. After incubation, the samples were centrifuged for 5 min, and then 1 ml of supernatant was
transferred to a new 2 ml tube. Chloroform:isoamyl alcohol (1 ml) (CIA) was then added to each
tube and mixed by inversion 15 times. All tubes were then centrifuged for 1 min with two 500 µl
aliquots of supernatant from each tube then transferred to two new 1.5 ml tubes. Cold ≥ 96% EtOH
(1 ml) is then added to each tube together with 50 µl of 7.5M ammonium acetate. The tubes were
then centrifuged for 20 minutes. At this point each tube had two clear phases. One large phase on
top and one small phase with a more viscous consistency at the bottom. 800 µl of the upper phase
was removed and replaced by an equal amount of 70% EtOH. After further centrifugation (15 min),
the DNA formed pellets. These were washed with 70% EtOH before being dried. Each pellet was
then re-suspended in 60 µl 10 mM TRIS (pH 8).
HYB_F120
CTGCCGTCATGTCTAGTTTTGG
HYB_R635
GAAAGAGCCCATATGGAACACC
MDH_F1
GCTCCTGTGGAAGAGACCC
MDH_R1
GCTCCAGAGATGACCAAAC
Table 2: The primers used in this study.
All of the samples were tested in a standard PCR procedure based on QIAGEN's HotStartTaq set.
The main primers are the HYB F120 and R635 and the MDH F1 and R1. Other primers were tested
but were not used for any of the sequences in this paper. Most of the Aurantioideae samples resulted
in positive bands when run in an 1% Fermentas agarose gel. A few of the other samples tested
resulted in positive agarose bands. Most of them, however, did not. Both 10% dilution and
increased concentration of the template DNA were tested without success. Samples that gave faint
but visible bands in the standard PCR gave strong bands when rerun another 30 cycles with new
dNTP and MgCl2. This also resulted in secondary bands, making gel separation necessary for
further sequencing of the genes.
Temperature Gradient Gel Electrophoresis (TGGE)
There were notable levels of polymorphisms and occasional length variations in the HYB genes
sampled. The TGGE method, described by (Myers et al., 1985) and developed for separating
paralogues of single copy genes in polyploid species by (Töpel, 2010), was used to identify the gene
variants. Special GC-rich anchor primers were developed and DNA that included a GC tail in one
end were made. The first gel, designed to identify a suitable melting temperature used Afraegle
DNA. It was determined that 34-39C would be a good temperature range. However, the first
parallel gel where this temperature range was used resulted in most of the sampled species ending
outside of their ideal melting range. A second parallel gel which had a 31-37C melting temperature
range gave good bands for all sampled specimens. The bands were cut out of the gel using fresh,
sterile scalpel blades. New blades were used for each new gel slice. The gel slices were put in PCR
tubes together with 50 μl of TE. Then the tubes were run in a PCR for 95C 20min to release the
DNA from the gel. The tubes were then used as source for new PCR runs where the original primer
was used on the side of the GC-primer and an inner primer was used in the other end.
The sequencing of standard PCR products was performed by Macrogen and the individual
sequences were then assembled with BioEdit (Hall, T.A. 1999). Most (22/27) of the sequences had
polymorphisms. Polymorphic sequences needed to be separated into the correct allelic or copy
phase in order to analyse them. As we were not successful with the TGGE method, instead we
assigned polymorphisms detected by overlapping peaks in the sequence trace files as follows. The
alleles of species previously inferred to be sister for cpDNA regions (Bayer et al., 2009) were
compared with those polymorphic sequences to be phased, on the assumption that the gene
sequences would share similarities in the nDNA regions used here. Then, all polymorphisms shared
with the sister sample were assigned to one allele in the polymorphic sequence. In the case of
Balsamocitrus, this resulted in one of its alleles being more closely related to Aegle than the other,
although in all other cases the inferred alleles for an individual were sister in all analyses. This
assumption should have no topological affect in phylogenetic inference (except for Balsamocitrus),
although a slight over or underestimation of terminal branch lengths might occur. The number of
polymorphic sites within an individual's sequence ranged from nil to 20, which is generally low
relative to the number of p.i. sites across each gene. Clearly there is much uncertainty in this
assumption as the paralogues/alleles may have split form a common ancestor allele, where both are
now separately evolving in diverging directions.
The sequences were then aligned to each other, starting with a MUSCLE alignment followed by
manual editing in BioEdit and Geneious (Drummond AJ, 2010). Parts of sequences for certain
species, mainly non-Aurantioid Melicope and Philotheca, which could not be reasonably aligned
with the main body of species, were instead off-sett to avoid non-homologous comparisons.
DATA Analysis
MrBayes
The samples that were successfully sequenced and
1 12B_koenigii
13B_koenigii
aligned were pooled with previous sequences
119Clausena
18Clausena
26M_paniculata
1
prepared by (Ramadugu et al., in prep.). Chloroplast
20M_caloxylon
121M_caloxylon
30P_missionis
sequences by Bayer et al (2009) were downloaded at
129P_missionis
0,77
1
1
1 31P_lobata
TreeBASE and paired with the nuclear gene data.
32P_lobata
0,99
5A_paniculata
0,79 4A_chevalieri
MDH and HYB alignments were prepared with
1 3A_chevalieri
1
11B_dawei
0,98
Muscle. One set of alignments was further edited
1
10B_dawei
1 2A_marmelos
11A_marmelos
manually for a decrease in off-setting. Thus, two
124M_cornuta
25M_cornuta
alignments for each gene were tested for models
137S_glutinosa
1
38S_glutinosa
using ModelTest in PAUP and the windows version
127N_crenulata
28N_crenulata
1
8A_monophylla
of Modeltest3.7 (Posada D and Crandall KA 1998).
19A_monophylla
0,96 1
17A_ceylonica
The alignment files were then prepared for MrBayes
6A_ceylonica
35P_trifoliata
0,91
(Huelsenbeck, J. P. and F. Ronquist. 2001, Ronquist,
1 33P_trifoliata
36P_trifoliata
34P_trifoliata
F. and J. P. Huelsenbeck. 2003). Modeltest
1
17C_sinensis
116C_sinensis
suggested TVM + Gamma as the best model for
0,56
114C_gracilis
15C_gracilis
1 23M_australasica
both genes and all four alignments. This model is
1 22M_australasica
intermediate in complexity between GTR and HKY
0.03
in MrBayes. Files were therefore made to run
Figure 1: The MrBayes tree of the HYB alignment.
MrBayes on both GTR + Gamma and HKY +
The genera are: Bergera, Clausena, Murraya,
Gamma models. MrBayes consensus trees were
Merrillia, Pamburus, Paramignya, Afraegle, Aeglopsis,
made using Toona as out-group (as per Bayer et al., Balsamocitrus, Aegle, Monanthocitrus, Swinglea,
2009) for the MDH gene tree and Clausena as out- Naringi, Atalantia, Poncirus, Citrus and Microcitrus.
group for the HYB tree. While Clausena is part of
the Aurantioid in-group in question, it has been found at its edges and were therefore deemed to be
suitable as an out-group. Not specifying any out-groups would have made comparison between the
two trees much more challenging.
Tests for intra-genic incongruence
The trees inferred for each gene were not exactly the same. As there can be multiple causes of
incongruence, several methods were used with the nuclear genes to examine possible causes.
Recombination within the sequences alignments would skew the tree-making efforts such as the one
by *BEAST. Therefore the RDP (Recombination detection program)(Martin DP, Lemey P, et al
2010) program was run using all available scanning methods (they are listed in the bibliography) to
identify potential recombination events. For the Bootscan and SiScan methods, different window
sizes such as 150, 200, and 250 were tried.
Positive selection could affect phylogenetic inference via convergence at non-synonymous sites. To
identify potential cases of positive selection the data was examined through the Phylogenetic
Analysis by Maximum Likelihood (PAML)(Yang, Z. 2007) program package. Since PAML
measures the ratio of nucleotide changes that affect amino acid changes, it requires that the
alignments are in codon order. The easiest way to do this is to reduce the alignments to exon-only
datasets. With the HYB data, three of the species (Atalantia ceylonica, Bergera and Paramignya
lobata) had insufficient length and had to be removed for this test.
For the MDH data, the two Skimmia alleles were
too short and removed. The yn00 program (Yang
& Nielsen 2000) was used to “estimate
synonymous and non-synonymous substitution
rates (dS and dN) in pairwise comparisons of
protein-coding DNA sequences”, and the results
were examined. Some species from the MDH
gene alignment were further examined in
SplitsTree (D.H. Huson and D. Bryant, 2006),
where the network was made using the full
sequence and compared with the exon-only
network. The later was examined as a whole and
also using only 1st and 2nd codons versus 3rd
codons.
Hybridization tests
The last search for sources of incongruence
involved finding evidence for possible
hybridization. The method described by
(Maureira-Butler et al., 2008) was used. This and
the following analysis were done on only the
Aurantioideae clade of Rutaceae. Several of the
non-Aurantioide alignments had proven to be of a
bad quality or too short in the previous tests.
They were also generally difficult to align with
the majority Aurantioid sequences and were
therefore trimmed from the MDH and CP trees
before running it on BEAST 1.6.1. (Drummond
AJ, et. al., 2002, Drummond AJ & Rambaut A,
2007) A GTR gamma model with a relaxed
lognormal clock (Drummond AJ, et. al., 2006)
and a UPGMA, Yule process speciation tree prior
was used. The root node was calibrated to 19.8
MA (12.1-28.2) after Pfeil and Crisp (2009).
However, it was found that the posterior and
likelihood values soon turned into infinity. A new
file was therefore prepared and run with the
uncorrelated log-normal relaxed molecular clock
(ucld) mean upper and lower borders fixed to
between 0.1 and 0.000001 (equivalent to a clock
rate of 10-7 to 10-12 substitutions per site per
year).
0,63
1
0,55
0,63
0,39
0,66
12C_ternata
52T_ciliata
49S_anquetilia
50S_anquetilia
56Z_monophyllum
55Z_monophyllum
1
47R_graveolens
26F_australis
33M_minutum
0,99
9B_koenigii
1
10B_koenigii
0,18
20C_excavata
1
21C_harmandiana
0,44
29G_trichanthera
1
27G_mauritiana
1
28G_mauritiana
1
30M_caloxylon
1
35M_paniculata
39P_missionis
0,81 40P_lobata
1
1
41P_scandens
53T_trifolia
1 54W_dolichophylla
1
1
34M_cornuta
3A_paniculata
1
2A_chevalieri
0,86 1 1A_marmelos
0,99
8B_dawei
1
7B_dawei
42P_latialatum
0,78
11B_malaccensis
0,32
0,99
24F_limonia
0,64
51S_glutinosa
36N_crenulata
0,98 13C_daweana
0,87 1 14C_schweinfurthii
0,99
48S_buxifolia
1
4A_ceylonica
1 6A_monophylla
1
5A_citroides
0,97 46P_trifoliata
44P_trifoliata
1
43P_trifoliata
45P_trifoliata
1 23E_glauca
0,08 22C_polyandra
1
38O_sp_nov
0,48
32M_papuana
0,1
17C_gracilis
0,02
31M_australasica
0,02
37O_neocaledonica
0,09
15C_amboiensis
0,99
16C_amboiensis
1 18C_sinensis
119C_sinensis
1
25F_oblata
Twenty trees from the end of the MDH and CP
0.03
posterior distributions of trees were taken for
Figure 2: MrBayes tree of the MDH alignment. The
further analysis (although taking only every 5th genera are: Toona, Skimmia, Choisya, Zanthoxylum, Ruta,
tree to minimize dependency between them).
Flindersia, Micromelum, Bergera, Clausena, Glycosmis,
These trees were then imported into Mesquite
Merrillia, Murraya, Pamburus, Paramignya, Triphasia,
where the time scale was converted to the numberWenzelia, Monanthocitrus, Afraegle, Aeglopsis, Aegle,
Balsamocitrus, Pleisopermium, Burkillanthus, Feronia,
of generations. Two sets of files were made,
Swinglea, Naringi, Citropsis, Severinia, Atalantia,
where one set of files was calculated to a 5 year Poncirus, Eremocitrus, Clymenia, Oxanthera, Microcitrus,
generation time and a 4 000 individual population Citrus, Microcitrus, Oxanthera, Citrus and Feroniella.
size. Another set was calculated to a 50 year generation time with a 40 000 individual population
size. Thereafter each of the 20 trees was used as a surrogate species tree for a simulation of 20 new
trees using a coalescent model where linage sorting alone can produce differences among simulated
gene trees.
The real generation time and historical population sizes were unknown, and it was assumed that
species representing 40 different genera would have a wide range of both generation times and
historical population sizes. Therefore both the lower and higher ranges were examined. The
distances between each of the trees chosen from BEAST was then compared with its 20 simulated
trees using *PAUPs TreeDist command in order to construct null distributions. Each of the CP trees
was also measured against each of the MDH trees – these distances are the observed distances
between the gene trees accounting for uncertainty in their inference. The test of Maureira-Butler et
al. (2008) compares these distributions to determine whether the null of lineage sorting can be
rejected to explain gene tree incongruence.
We assumed a generation time of 20 years, based on observations of the time to maturation in
Citrus, although this is a somewhat arbitrary value because of the occurrence of clonal reproduction
(via nucellar embryony) in some species (M.L. Roose, pers. com.). Further, the mean ancestral
effective population sizes of several Citrus species have been estimated to be around 4,000 - 4,500,
based on the diversity found within three nuclear genes (Ramadugu et al., in prep.).
Therefore, a simulation set with these characteristics (20 years and 4 000 population size) was
prepared and compared with the previous results. The 20 year generation time was also used with
the 40 000 population size.
Thereafter, ten species were chosen which had been incongruently placed when comparing the CP
and MDH trees generated by either BEAST or MrBayes. These were removed from the lower range
tree distance analysis one at a time and their individual effect on the test statistic determined.
To account for the uncertainty in the data, the 95% credibility interval for the CP/MDH data test
statistic was obtained through discarding the ten highest and ten lowest numbers. The MDH
simulated trees null distribution was closer to the observed distances than the CP null distribution
and therefore produces a lower type 1 error. We used this null in the following calculations. The
highest values of the 20/4 000 and the 20/40 000 distances for simulated trees for MDH were
compared with the lowest remaining value of the CP/MDH distances. This resulted in two numbers
for each species deletion set. These numbers were compared with the results where no deletions had
taken place and three of the species ended up with a 4 point difference. A further three of them had
a 2 point difference, whereas the remaining four tested had less affect on the test statistic and were
not considered further. These six species having the largest affect (Bergera koenigii, Oxanthera sp.,
Feroniella oblata, Severinia buxifolia, Swinglea glutinosa, and Clymenia polyandra) were
identified for combined deletion tests. They were tested in several deletion series where they were
removed one at a time, adding one removal to the preceding one until all six had been removed
from the full alignment. This was done for the 20/4 000, 20/40 000 simulation sets and several
combinations of removal order were tried. The size of distance reduction between the simulations
and the original trees remained the same as in the first test. Although we cannot be certain, the first
three species may be of hybrid origin and were therefore excluded from the *BEAST tree-building
analysis, because this analysis assumes no hybridisation.
*BEAST
Two *BEAST (Joseph Heled and Alexei J. 2010) runs were performed. The first used all chloroplast
and MDH sequences for Aurantioideae that we had available except the three mentioned in the
previous analysis. The second used chloroplast, MDH and HYB sequences for all Aurantioideae
species that were represented at least once by all three genes. Again, Bergera koenigii,the only
probable hybrid species that was available for HYB, was excluded here.
The *BEAST set up file for the CP/MDH set up assumed a coalescent start tree, which is the default
option, for CP while using a UPGMA start tree for MDH. All trees in the three gene set up assumed
UPGMA trees. The CP data used a modified mitochondrial ploidy level, where the original value of
0.5 was changed into 1 to account for the assumption that these plants are all hermaphroditic and
thus may inherit the plastid from either parent. The CP/MDH *BEAST file was divided in four files
that each proceeded for 30 million generations, whereas the CP/MDH/HYB *BEAST file was
divided in three files each doing 40 million generations, thus producing a total of 120 million
generations for both sets.
The resulting trees files were examined in Tracer. Excluding a 10% burn-in, the trees generated by
each *BEAST run were merged to files covering the full analysis. Maximum clade credibility trees
were then made using TreeAnnotator v1.6.1
Lagrange Geographic Range Evolution
The species trees made by *BEAST were turned into NEWICK format and used as a base in a
Lagrange biogeography analysis. Both the 2 gene tree and the 3 gene tree were used as a base for a
Afr1
Southern and Western Africa
af1 af2 ind pam soa sin aus ncd pap
Afr2
Eastern Africa
af1
Ind
India
af2
Pam
Pamir (including the Indian provinces bordering the
Himalayas from Nepal and west)
ind
SOA South-East Asia (Burma and east)
pam
Sin
China
soa
Aus
Australia
sin
NCD New Caledonia
aus
Pap
Papua (coding for all islands between Malaysia and the
Australian mainland)
ncd
Svh
The Pacific Islands
pap
—
1
—
1
1
—
1
1
—
1
1
—
1
1
—
1
—
1
1
—
1
—
Table 3: The Lagrange region codes followed by a table showing which areas were coded as
neighbours to each other.
Lagrange biogeography. Besides the dated Newick tree, Lagrange uses a species matrix text file as
input. A 10 region master matrix was made including the regions in [Table3]. The matrices used for
the different species configurations were then adopted from this list. Lagrange further requests
information on which regions neighbour each other.
Lastly, Lagrange requests information on the likelihood of dispersal between the different areas.
Two different examinations were made for each species tree. The first limited the amount of areas
as suggested by the Lagrange Configurator coding program. This run also assumed a dispersal
probability of 1 for neighbouring regions and a probability of 0.5 for all other areas.
Range limitations for the 3 gene tree included removing Ncd and Svh which had no representatives
among the species in this tree. Further, the Pam region was also excluded. These species were also
present in India. The second round included the Pam region. For the first 2 gene tree, all of Africa
was made into one region, India and Pamir were joined, Australia and New Caledonia were joined
and Papua and the Pacific Islands were joined to each other. The second 2 gene tree analysis used
the regions found in the neighbouring regions table.
The second round of analysis also differed from the first in a more scaled dispersal probability.
Neighbouring regions still had a probability of 1, but now a dispersal that jumped over one region
had a probability of 0.7 and one jumping over 2 regions a probability of 0.4. Jumping further than
that had a probability of 0.1, except if the jump took place over land in the 2 gene case when the
dispersal probability was set to 0. Practically, this meant that nothing was regarded as likely to
disperse directly between China and Africa, between Australia and Pamir or between New
Caledonia and any area except Australia, Papua or China.
BEAST Phylogeography
A phylogeographic analysis was run using BEAST (Lemey P, Rambaut A, Drummond AJ &
Suchard MA, 2009). For the BEAST phylogeographic analysis, the A-G rate in the GTR model
fixed to "1" through unchecking it as an operator in BEAUTi, thus remaking it into the TVM model.
Initial attempts to have a working BEAST phylogeny linked the MDH and CP dataset results.
Doing so consistently resulted in unconverged data for the prior, posterior, Yule-birthrate, clock
rates and tree probability. Therefore the two datasets were examined unlinked, producing separate
output. This analysis was also run with the three possibly hybridised species removed.
Two different biogeography model runs were set up. The first was a discrete phylogeographic
analysis, which used area code names. The coded areas were:
ASIA_TROP_EAST Southeast Asia
ASIA_TROP_WEST Southwest Asia
ASIA_TROP
Southern Asia
AFRICA_W_T
Western Tropical Africa
AFRICA_E_T
Eastern Tropical Africa
AFRICA
All of Africa
ASIA
All of Asia
PAPUA
Islands between Australia and Malaysia
AUSTRALIA
Australia
CHINA
China
NEW_CAL
New Caledonia
The other was a continuous phlyogeographic analysis that used latitude and longitude. These were
chosen from the centre of each species' distribution area as identified through applying the GRIN
native distribution range on Google Maps.
The different coding used in BEAST compared with Lagrange are due to the requirements of each
program, where Lagrange can accept one species inhabiting several different areas whereas BEAST
can not.
RESULTS
The species table [Table 5]show all species that have been a
part of this study through either sequences, PCR or extraction.
They represent all subfamilies and tribes in the GRIN
classification and all Englers subfamilies except Spathelioideae.
Several further species were also acquired although no work
was done with them. They also cover 22 of Englers 23 tribes.
Therefore the potential coverage of the Rutaceae has been
Figure 3: Electrophoresis gels, with
the Ext24 gel to the left and the Ext25 gel
good.
to the right. The Ext24 gel has two
ladders, where the upper one is a
GeneRuler High Range 10000-48500 bp
and the lower is a FastRuler Middle
Range 100-5000 bp ladder. Ext25 only
has the High range ladder, the well at its
bottom.
Sample Processing
A total of twelve extractions were done in this course, see [Table 4].
Omega kit(1) The first (Ext23) extracted from thirteen silica gel dried species specimens and nine
of them resulted in successful PCR reactions. The second extraction (Ext24) had 12 herbarium
specimens from an equal amount of species. One of the samples resulted in a successful PCR
amplification.
CTAB I (2) Ext25 included 11 samples where the first four were fresh
silica gel dried whereas the remaining seven samples were from the
herbarium. All of these samples had 50% diluted extracts quantified
measuring the 260/280 nm absorbance ratio and DNA concentration
using a Pharmacia Genequant II. The ratio was generally between 1.75
and 1.86 with concentrations between 65 and 125. One sample had a
concentration of 5 with a 1.75 ratio while another sample had a
concentration of 170 with a ratio of 1.57. Electrophoresis (due to
inexperience, all electrophoresis results presented here on extractions
were performed after Ext27 and were thus unknown during the larger
part of this process) showed that the fresh leaf samples and one
herbarium sample were likely candidates for successful PCR. However,
only two of the fresh samples gave weak positive results in the PCR.
Figure 4: Ext26, it has
CTAB II (3) First among the extractions exploring the Scott and
both the high and middle
Playford extraction protocol (Ext26) used 18 samples, where 17 were
silica gel dried and one was a herbarium specimen. Most of the pellets range ladders as described
above.
were characterised by being transparent and having a gelatinous
appearance. Nine of them gave 260/280 absorbance results between 1.55 and 1.92. The lowest
measured result was 1 and the highest was 6.99. Electrophoresis suggested that only three of the
samples might give positive results and no successful PCR results were produced.
Ext26b used six samples, where five gave gelatinous pellets. The 260/280 absorbance results ranged
between 1.35 and 2.5 and the electrophoresis results suggests that only one (sample 4) of the
samples had potentially extractable DNA. However, a PCR performed to test new primers gave
positive results for sample 3.
In Ext26c, two of the samples produced gelatinous pellets while the rest had standard pellets. The
electrophoresis show better results for the sand grinding compared with the N2 grinding extractions
and all samples except (sample 1) result in bands and only (sample 2) resulted in amplification in a
PCR testing the sand extractions.
Figure 5: Extractions Ext26b, Ext26c, Ext26d and Ext26f. Ext 26b has a high range ladder while the other gels have
both high and middle range ladders.
Ext26d showed that repeating the extraction buffer step reduced yields dramatically (absorbance
concentration reductions were as follows: 184 → 33; 175 → 85). The second species testing double
extraction buffers had used two different leafs for the different samples, where one leaf was young
whereas the other was mature. The mature leaf had a markedly higher viscosity in the extraction
buffer compared with the young leaf. Absorbance ratios ranged between 1 and 8.2 with a median of
2.1. Three of the samples (3,4,5), which formed nice pellets after one EtOH precipitation resulted in
good electrophoresis bands. The other samples had a repeated precipitation step and produced either
very weak bands (1,2) or had most of its contents remain in the well (6). Only one (1) of the
samples produced a weak result in PCR.
Due to the extra washing step after the samples had dried over night, no results were gained for the
Ext26e extraction. The Ext26f extraction resulted in blurry electrophoresis bands for the four
samples (1,3,4,5) which produced pellets. No sample resulted in successful PCR. The EtOH
experiment in Ext27 resulted in only the supernatant samples showing evidence of DNA in the
electrophoresis test. No PCR was performed. Ext28 showed that only taking the middle part of the
supernatant after CIA cleaning produces cleaner electrophoresis bands than the end parts do. These
results failed to transfer to the PCR test. The final extraction (Ext29) resulted in long and blurry
electrophoresis bands for most species (3,5,8 being exceptions). The samples for specimens 2 and 4
also produced bands in the EtOH wash. All samples also had their absorbance ratios examined, and
no resulting ratio was close to indicating DNA. Not surprisingly, the PCR gave depressing results
both before and after a QIAEX II desalting protocol had been used on the samples.
Figure 6: Ext27, Ext28 with its "a" phase to the left followed by the "b" phase, Ext29 with the "a" phase to the left
followed by the "b" phase and the EtOH wash phase. Ext27 has only the high range ladder, Ext29 EtOH has only the
middle range ladder and the others have both ladders.
Extraction
Ext23
Method used
Omega
kit
Omega
kit
CTAB
CTAB
S&P
Nr of extracted samples
13
12
11
18
PCR result
10
77
1
8
3
27
0
0
2
33
1
13
-
-
2
17
5
45
4
22
1
17
7
88
electrophoresis result
% Ext24
% Ext25 % Ext26 % Ext26b % Ext26c %
CTAB
S&P
6
Extraction
Ext26d % Ext26f % Ext27 % Ext28 % Ext29
Method used
CTAB
S&P
CTAB
S&P
6
CTAB
S&P
CTAB
S&P
8
12
CTAB
S&P
8
%
CTAB
S&P
Nr of extracted samples
6
11
PCR result
1
17
3
50
0
0
0
0
1
9
electrophoresis result
5
83
4
67
3
38
10
83
7
64
Table 4: The extractions made during this project are listed
together with their success rate. Ext26e is not listed since no
results were obtained from it.
TGGE results
The first parallel gel resulted in inconclusive results. Two
specimens, Afraegle (6), counting from the left, and Philotheca
(8) found melting points where they could stop in the gel. The
others did apparently melt already in the well, and therefore
Figure 7: TGGE gel 1
migrated straight out of it. The second parallel gel gave a more even result, showing that almost all
species melt within a fairly short temperature range. The bands were generally close to each other,
but some of them could be distinguished and cut out for extraction testing. These were Dinosperma
(9), Choisya (11), Merrillia (12), Atalantia (13), Paramignya (15), Bergera (16), and Murraya
(17). All species except Atalantia and Bergera contributed two bands.
Several attempts to recover DNA from the gel slices failed and
further work on the method was aborted. One possible reason
for this that was not explored further was the realisation that the
outer and inner primers were located next to each other with no
spacing between them. It is also possible that a favourable result
would have been obtained through using overlapping inner
primers, thus increasing recovery of possibly broken DNA
fragments. With a reliable solution for DNA extraction from the
gel, this method is expected to be a fast and accurate competitor
to paralogue separation by cloning.
Figure 8: TGGE gel 2
DATA Analysis MrBayes
Modeltesting
Comparing the different genes that were available resulted in 47 species that were represented by
both the chloroplast data and the MDH gene. A total of 20 species were found for the combination
of chloroplast, MDH and HYB sequences. Comparing the four trees resulting from running
MrBayes with both the GTR and HKY models on both sets of alignments gave no significant
differences, although the consensus tree based on the GTR model using the manual alignment had
stronger support in some nodes than other combinations of model and alignment.
Recombination testing using RDP
Running the HYB file in RDP did not find any support for recombination.
Running the MDH file in RDP resulted in a single putative recombination event for Skimmia.
However, the event was at the end of the sequence which was of a poor quality and that is the likely
cause of the signal, rather than true recombination. Due to its poor quality, the sequence was
removed from further analysis.
Positive selection testing using PAML
In HYB, one possible case had an omega value of 0.86 (values higher than 1 are usually required at
specific sites, but averaged across a sequence values lower than 1 found using Yang and Nielsen
(2000) indicate sequences to investigate further) suggesting that positive selected for evolution was
found between one allele of Citrus sinensis and one allele of Clausena hamandiana. The C.
hamandiana sequence differed from the Citrus in two nucleotide locations compared with the other
C. hamandiana allele. Upon further examination in SplitsTree, it was discovered that the respective
alleles stick together within the genus and that the alleles from each genera are always far from each
other in the network.
In MDH, 110 cases had results with an omega number between 0.5 and 99, which identifies
possible positively selected evolution. The smallest common denominator summing up these 110
cases involved the following species; Burkillanthus, one of the Glycosmis mauritiana alleles,
Paramignya lobata and Paramignya scandens, Severinia buxifolia and Swinglea glutinosa
combined with each other or other species.
The two Glycosmis mauritiana alleles were always found together in the phylogeny and forming a
clade with Glycosmis trichanthera. This is fully expected given the taxon sample. The two
Paramignya species were consistently found together in the full sequence network as well as with
the exon, 1-2 codon network. Paramignya scandens also had a clear relationship to Burkillanthus in
the exon 1-2 codon SplitsTree network, which is inconsistent with the MrBayes tree structure.
Further investigation of the sequence alignment revealed that this effect was caused by a single base
that Paramignya scandens shared with Burkillanthus but not with Paramignya lobata. This relation
between Burkillanthus and Paramignya scandens was the only one to consistently appear with
Burkillanthus through all the SplitsTree network constellations that were examined. Severinia was
consistently grouped with Atalantia and Citropsis in the SplitsTree network as was expected.
Swinglea did not share any noteworthy splits with other species. After thus examining the full
sequences and comparing them to first and second codon positions while finding no significant
differences, it was concluded that any positive selection leaves the phylogenetic signal of the full
sequence unaltered. The concerns raised by PAML where therefore not corroborated.
Hybridization test results
The simulation test results showed that at the larger generation time and population size
combinations (50/40 000), the null hypothesis of lineage sorting alone could not be rejected.
However, at every other lower value combination (20/40 000 and smaller) the null hypothesis could
be rejected for three or more samples. When the individual effect on the test statistic were
examined, three species were found to have the greatest and consistent effect, with no difference in
removal order (Bergera koenigii, Oxanthera sp. and Feroniella oblata).
*BEAST tree results
Two species trees were produced. One was based on 37 species, the three species identified in the
hybridisation test having been removed, using the CP and MDH data (figure 10). The other was
based on 18 species and CP, MDH and HYB data (figure 9). The two trees had different root ages:
the 3 gene tree had a root age of 15.24 MA, whereas the 2 gene tree had a root age of 10.55 MA.
Neither of the trees had a root age prior, and differed only by starting tree modelling.
Lagrange Biogeographic results
Both the simple and advanced 3 gene trees gave the same results except on the N3 node (see table
7). The N4 node, which includes Clausena, Merrillia and Murraya and the root node: N34 were
regarded of Papuan origin. The N26 node, forming a clade with Afraegle, Aeglopsis, Balsamocitrus
and Aegle were regarded African/African-Asian whereas all other nodes were inferred to have most
likely been present in India.
The two 2 gene trees disagreed on the location of the higher nodes (N72, N70, N69, N66, N65,
N62, N61) (see table 6). The simple model favoured Papua as the origin of the root and higher
nodes, whereas the more advanced model favoured South-east Asia. At the N37 and N27 nodes, the
advanced model has India-Papua and India as primary choices for branch radiation whereas the
simple model in both cases instead inferred South-east Asia. The results of the advanced models are
included in tables 6 and 7 with the related cladograms.
BEAST biogeography
BEAST produced separate trees for the CP and MDH trees. The results support a south-east Asian
origin for the Aurantioids. It gives a Papuan origin for the Citrus species, but almost all other
development in the higher nodes take place in south-east Asia. The two trees differ regarding the
node that gathers the Balsamocitrinae clade, where the MDH results have these developments take
place in Africa while the chloroplast tree place them as well in south-east Asia. Removing the three
possibly hybridized species made convergence possible.
Discussion
Extractions
The first lesson from my extraction experiments is the importance of evaluating results after each
extraction. Up to Ext26e, results were becoming better with the level of polysaccharides decreasing
with pellets no longer exhibiting the gelatinous properties from the first extraction attempts and
with the EtOH precipitation working fairly smoothly. If my hindsight is correct, and I did see dried
DNA which I interpreted as dried salt, my search for a working extraction protocol could have been
successfully accomplished here. Lacking any verifiable results on this extraction, it is none the less
clear that the following extraction (Ext26f) produced the best PCR result (for Ruta pinnata) gained
through the CTAB extractions. The Ruta pinnata PCR was equal to the results gained for Ruta
graveolens after the kit extraction, and the CTAB further resulted in weaker bands for both
Phellodendron species that had not extracted with the kit. Ext26b gave an almost perfect result in
both PCR and absorbance ratios for Phellodendron amurense, although the gelatinous pellets both
this and the other samples had were problematic. The tubes from this extraction all ended with a
several millimetre thick gel-like sedimentation in the bottom. Therefore, much work and possibly
better results would have been gained by running extractions on electrophoresis earlier and to
amplify the extracts more faithfully and not least taking the time to step back and consider the work
from a distance once in a while.
The second lesson learned was the value of understanding how the processes used work and what
the ingredients do. Knowing what an added ingredient is intended to do is crucial in understanding
how changing its recipe might affect the end results. Both realising the effect of NaCl concentration
for reducing polysaccharides contamination and the importance of reaching a 75% end
concentration at the EtOH step were important lessons. The negative impact of sub-zero
temperatures on Rutaceae extraction is another lesson which might have provided better result if the
evidence had been noticed earlier. The extraction notes mention the thick extraction consistency but
fails to put it in relation to the cold until a mention of it was made during an email conversation
with K. Scott.
It is also unclear what the relationship between extraction results and PCR results was. Several of
the non-Aurantioid species resisted amplification when extracts made by others, which had been
successfully used in chloroplast based studies, were used as PCR templates for low-copy nuclear
genes. It is therefore possible that the main problem does not rest with the extractions once the
above mentioned extraction issues have been dealt with, but with the primers used. It seems that
finding new primers is relatively difficult for species that have not had extensive sequencing done
previously, and that primer identification for the non-Aurantioid species therefore has been out of
reach for this project. I believe that finishing the goal of inferring a phylogeny of the Rutaceae using
low-copy nuclear genes will require that this genome-fishing takes place for perhaps 4 or 5 species
throughout the remainder of the family.
Classifications
The 3 gene tree results from *BEAST do not support the Balsamocitrinae / Citreae division found in
the GRIN classification. Instead it takes Monanthocitrus, Pamburus and Paramignya and moves
them into the Balsamocitrinae subtribe. Engler, who had a slightly different classification of the
Aurantioids, has a similar problem with Monanthocitrus moving from his Hesperethusinae subtribe
into Citrinae. The second large inner clade in the 3 gene tree suggests further breakdown of the
Citrinae subtribe.
The situation is not helped by the larger number of species in the 2 gene tree. The GRIN tribe
Clauseneae and the subtribe Merrilliinae fail to form clades but instead form a grade relative to the
root node. It further supports the 3 gene tree in moving genera to the Balsamocitrinae subtribe and
further adds Triphasia and Wenzelia to it. It further divides the Citreae into a clade containing
Citrus proper (e.g. Citrus, Microcitrus, Poncirus, Clymenia and Oxanthera) and a clade for the
remaining species. Compared to the Engler subtribes, Citrinae is now evenly divided between the
three clades mentioned whereas Hesperethusinae is divided between the two later clades.
Comparing the 2 gene tree with Samuel's (2001) atpB/rbcL tree in Fig 1, the Clauseneae tribe
proposed fails to form a clade. The Citreae tribe changes places between Paramignya and Citropsis
and lifts out the Atalantia/Severinia clade altogether compared with my results.
There is better agreement with Chase (1999) in Fig 3 where the placement of Clausena next to
Aegle is the main problem.
Besides the clade gathering Citrus, Clymenia, Microcitrus and Poncirus together, there is no
agreement between the 2 gene tree and Morton's (2003) rps16/trnL-trnF trees. The situation is more
complex in the 2009 paper. Here several trees are published, starting with a trnL-trnF tree that says
little more than that nodes (see cladogram 2) N16 and N27 exist. The second tree, based on rps16, is
a little more detailed, giving some resolution for the N51 clade, but inexplicably joining Clausena
to it. Her third tree is based on atpB-rbcL and largely has the same topology as the previous trees,
with the exception of Murraya ending up sister to Pamburus. This is an anomaly that will be
discussed further. The fourth tree is based on ITS and is also generally in agreement with the
previous trees, which does not say much considering the very limited resolution of the
aforementioned trees. One important anomaly contained in the ITS tree is that Merrillia is placed
sister to Glycosmis.
The fifth tree presented in Morton (2009) is based on a concatenation of the previously mentioned
genes. The reason given for concatenating the data was the principle that if the individual gene trees
are not contradicting each other, there is no problem with joining the data. This requires that several
theoretical and methodological issues are ignored: This assumption forces you to disregard the
evolutionary differences between plastid and nuclear evolution, e.g. plastid and nuclear genomes
(and unlinked nuclear loci) would be separately affected by hybridisation and nuclear loci can be
paralogues. Further, plastid and nuclear genomes have different effective population sizes which
affects lineage sorting rates and thereby also coalescence times. The concatenated tree adds a little
resolution but retains the placements of Murraya with Pamburus and Merrillia with Glycosmis.
Finally the last tree in Morton (Fig. 6, 2009), which is based upon the previous data combined with
morphological data, we find that the resolution has increased and only the genera Wenzelia,
Merrillia and Murraya are conflicting with the results in this thesis. The pairing of Merrillia to
Glycosmis that was previously seen in the ITS (with a bootstrap support of 98) and concatenated
(91) trees, and of Murraya to Pamburus that occurred weakly in the atpB-rbcL tree (59 bootstrap
support) and hardly any better in the concatenated tree (61), now show an overwhelming support of
100 and 96. This is a huge increase for the latter. Morton (2009) concluded that neither the
Clauseneae nor Citreae tribes are monophyletic. The basis for Citreae failing to be monophyletic is
the Murraya-Pamburus connection, which hardly can be said to be supported without the
morphological data. The validity of her conclusion thus rests on whether concatenating molecular
data with morphological data is sound, or not.
Finally comparing with Bayer, with which some of the sequences and the method of analysis
(Bayesian analysis) is shared reveal that the placement of Eremocitrus glauca and added high-level
resolution are the only differences. The trees are otherwise in agreement regarding the clades.
Biogeography
The Citrus clade is divided into two parts, with most of the classic Citrus in one subclade N15 and
most of the new Citrus in another subclade N10 (see cladogram 2). The N15 clade gathers the
Chinese Poncirus, the Papua New Guinea Citrus “amboin” and Citrus sinensis of uncertain but
possibly Chinese origin. The N10 clade has 6 species out of which half are Australian. Another
species is from New Caledonia and the two remaining species occur in Papua New Guinea and the
nearby New Ireland island. The same results are found in the BEAST biogeographical analysis. As
all of the Papuan species in this group are found on the Australasian side of the Wallace line, this
clade is only found in Australasia.
(Beattie et al., 2008) suggested that the Citrus clade of the Aurantioids originated in Australasia,
which he defines as Australia, New Guinea and other smaller islands east as far as Fiji. He also
suggests that this is a more likely direction of dispersal, compared with migration southwards from
Asia, when the westward direction of ocean currents and terrane movements are considered (Hall,
1997). He may well be right concerning the ocean currents, but the dating of the Aurantioideae by
Pfeil (2009) using fossil evidence to calibrate a chloroplast phylogeny, as well as the dates gained
through *BEAST's analysis of the alignment here (informed by Pfeil's date calibration applied to
the species tree) this work suggests that the geological movements are so old that any island
movements hardly can be part of the explanation. As the Lagrange result table shows at N38 and
N16, the question regarding the origin of the Citrus clade is not whether it originated in Australasia
but how soon it started spreading north to China.
Despite the assumptions that must be made in Lagrange regarding probable dispersal routes and
choosing good geographical areas to correspond, it does none the less seem to be the better route to
take in this kind of study compared with the discrete BEAST phylogeography model. The reason is
that Lagrange accepts a species in several different areas, whereas BEAST does not. Thus the
confusing situation where a region such as Tropical_Asia equals a combination of two smaller
regions, Tropical_Asia_East and Tropical_Asia_West. All of these three regions are further part of
the general region Asia which also includes China. But about this, BEAST will know nothing and it
does therefore seem likely that its results can easily be confused on this regard. A continuous
BEAST phylogeography model including range-wide sampling with unique geographical
information for each sample would perhaps solve this problem. This would include gathering wild
samples from several parts of each species' range where more widespread species would require
more samples than species endemic to small islands. This would provide both geographic
information on the species area as well as possibly identify a smaller root node area, the place
where the species first developed, for each species. The collected data would also have to provide a
greater range of genetic information than is currently available.
Tables and Trees
Clausena_harmandiana
0,47
Murraya_paniculata
1
5,81
Merrillia_caloxylon
Swinglea_glutinosa
1
Naringi_crenulata
9,49
0,41
0,77
1
8,84
4,57
0,93
0,81
8,07
1
3,64
0,98
Citrus_gracilis
0,77
Afraegle_paniculata
1,52
Aeglopsis_chevalieri
2,38
0,96
0,28
12,11
11,33
0,87
0,42
6,4
7,95
0,66
0,98
1
Wenzelia_dolichophylla
0,18
Monanthocitrus_cornuta
Pamburus_missionis
1
5,76
2,09
0,99
1
2.0
Figure 9: The 3 gene *BEAST tree. The node labels
indicate age (in italics) and the branch labels indicate
posterior probability (in bold).
Citropsis_schweinfurthii
Citropsis_daweana
Swinglea_glutinosa
Burkillanthus_malaccensis
Pleisopermium_latialatum
Severinia_buxifolia
4,07
0,72
Atalantia_ceylonica
3,11
0,31
Paramignya_lobata
Pamburus_missionis
2,18
Feronia_limonia
5,18
0,98
6,03
9,7
Paramignya_scandens
Naringi_crenulata
3,81
0,97
0,11
5,64
0,63
5,2
0,36 0,71
4,6
5,9
0,43
Paramignya_lobata
Aegle_marmelos
1,65
Balsamocitrus_dawei
0,64
1,08 Afraegle_paniculata
0,57
0,58
Aeglopsis_chevalieri
1
0,99
7,05
0,63
Monanthocitrus_cornuta
0,3
0,96
Balsamocitrus_dawei
Aegle_marmelos
Murraya_paniculata
Triphasia_trifolia
5
Atalantia_monophylla
5,19
Merrillia_caloxylon
0,41
6,71
2,24
Atalantia_ceylonica
1
Clausena_excavata
3,28
0,98
9,11
Clausena_harmandiana
4,52
Microcitrus_australasica
1
12,56
Citrus_sinensis
Glycosmis_trichanthera
1
9,96
0,69
Glycosmis_mauritiana
5,63
10,55
Poncirus_trifoliata
15,24
0,9
Micromelum_minutum
1
12,51
1
Atalantia_monophylla
0,14
Atalantia_citroides
Poncirus_trifoliata
2,291
Citrus_sinensis
0,37
Citrus_amboiensis
2,77
Clymenia_polyandra
0,54
2,19
Oxanthera_neocaledonica
0,77
1,66
Microcitrus_australasica
0,58
1,39 Eremocitrus_glauca
0,39
1,21 Citrus_gracilis
0,54
0,85
Microcitrus_papuana
2.0
Figure 10: The 2 gene *BEAST tree. The node labels
indicate age (in italics) and the branch labels indicate
posterior probability (in bold).
Aegle marmelos (L.) Corrêa
H&M USDA PI 539142
Merrillia caloxylon Swingle
H&M USDA PI 539733
Aeglopsis chevalieri Swingle
H&M USDA PI 539143
Monanthocitrus cornuta Tanaka
H&M CANB TJ Hoe s.n.
Afraegle paniculata(Schumach.) Engl.
H&M USDA PI 103107
Murraya paniculata ( L.) Jack
H&M CANB 743224
Atalantia monophylla (L.) DC.
H&M USDA PI 109613
Pamburus missionis Swingle
H&M USDA PI 095350
Balsamocitrus dawei Stapf
H&M USDA PI 539147
Paramignya lobata Burkill
H&M USDA PI 600642
Bergera koenigii L.
H&M USDA PI 539745
Melicope elleryana ( F.Muell.)
T.G.Hartley
HYB PIF 34003 - 18/7/8
Citrus gracilis Mabb.
H&M CANB 644758
Philotheca deserti (E. Pritz.) P.G
Wilson var. deserti
HYB MJB 1919
Clausena harmandiana Pierre ex
Guillaumin
H&M USDA PI 600640
Melicope micrococca ( F.Muell. )
T.G.Hartley
MDH ANBG 8501279
Acronychia acidula F. Muell.
MDH ANBG c664755
Microcitrus australasica Swingle
MDH USDA PI 312872
Ailanthus altissima (Mill.) Swingle
MDH GotBot 1988-999
G, BRUNS
Micromelum minutum ( G.Forst.)
Wight & Arn.
MDH USDA PI 600637
Asterolasia hexapetala Druce
MDH ANBG c9505139
Murraya paniculata ( L.) Jack
MDH GotBot 1933-3691sG
Atalantia citroides Pierre ex
Guillaumin
MDH USDA PI 539145
Murraya paniculata ( L.) Jack
MDH Perth 853900 ((bot. g.
Perth))
Atalantia ceylanica (Arn.) Oliv.
MDH CRC 3287
Naringi crenulata ( Roxb.) Nicolson
MDH USDA PI539748
Bergera koenigiiL.
MDH CANB 743217
Nematolepis squamea ( Labill.) Paul
G.Wilson
MDH ANBG c629571-46
Boronia anemonifolia A.Cunn.
MDH ANBG 9506114
Orixa japonica Thunb.
MDH GotBot 1918-0012 G,
Hesse
Burkillanthus malaccensis (Ridl.)
Swingle
MDH CHM78–extraction
(CSIRO Plant
Industry)
Oxanthera neocaledonica Tanaka
MDH USDA PI 539671
Choisya ternata Kunth
MDH CANB 743233
Oxanthera Montrouz. sp
MDH Veillon 7758 ((sp. in
canberra))
Citropsis daweana Swingle & Kellerm. MDH USDA PI 247137
Paramignya scandens Craib
MDH USDA PI 109758
Citropsis schweinfurthii Swingle &
Kellerman
MDH USDA PI 231240
Phebalium squamulosum Vent.
MDH ANBG c653838
Citrus amboiensis (Citrus sp. “
Amboin, New Guinea ”)
MDH Merbein CO054
Pleiospermium latialatumSwingle
MDH USDA PI 600643
Citrus australis( Sweet ) Planch.
MDH Crisp 10432
Poncirus trifoliata ( L.) Raf.
MDH CRC 1717
Citrus glauca ( Lindl.) Burkill
MDH USDA PI 539717
Poncirus trifoliata( L.) Raf.
MDH CRC 3330
Citrus sinensis Osbeck
MDH CRC 1241
Ptelea trifoliata L.
MDH GotBot 1900-3888nU
Citrus wintersii Mabb.
MDH USDA PI 410943
Ruta graveolens L.
MDH GotBot 1997-3115 G,
SÄVE
Clausena excavataBurm.f.
MDH USDA PI 235419
Sarcomelicope simplicifolia( Endl. )
T.G.Hartley ssp
MDH ANBG 8501648
Clymenia polyandra ( Tanaka )
Swingle
MDH USDA PI 263640
Severinia buxifolia Ten.
MDH USDA PI 539793
Cneorum tricoccon L.
MDH 303GU Malaga,
Spain, 1968-04-16
SkimmiaThunb. sp.
MDH CHM75–extraction
(CSIRO Plant
Industry)
Correa decumbens F.Muell.
MDH GotBot 1986-1296
Lorentzon
*”Skimmia” (?) Unknown I.D.
MDH ”GotBot 1952-0046”
Dictamnus albus L.
MDH GotBot 1996Swinglea glutinosa Merr.
0506sw JJM950936
MDH USDA PI 142571
Erythrochiton brasiliensis Nees &
Mart.
MDH GotBot 20071167sG
Tetradium daniellii ( Benn.)
T.G.Hartley
MDH GotBot 2008-2718sW
Feronia limonia Swingle
MDH USDA PI236991
Toona ciliata M.Roem.
MDH 21B GH06-015
Feroniella oblataSwingle
MDH USDA PI 539720
Toona ciliata M.Roem.
MDH CANB 743215
Flindersia australis R.Br.
MDH CANB 743207
Triphasia trifolia P.Wilson
MDH USDA PI 539800
Glycosmis mauritiana Tanaka
MDH USDA PI 600641
Wenzelia dolichophylla Tanaka
MDH USDA PI 277441
Glycosmis pentaphylla Corrêa
MDH Merbein CR044
Zanthoxylum bungeanum .
MDH GotBot 2008-2762sW
Glycosmis trichanthera Guillaumin
MDH PI RRUT 12
Zieria tuberculata J.A.Armstr.
MDH ANBG c665919
Acradenia frankliniae Kippist
MJB 1964 - 29/4/8
Geijera parviflora Lindl.
ANBG 723469
Agathosma adenandriflora Schltr
GU 2613 P.A.Bean
S.W.Cape Ceres 1990
Geijera parviflora Lindl.
PIF 31159 – 27/3/8
Amyris elemifera L
2007-0711 A Fairchild
Halfordia kendack ( Montr. )
Guillaumin
PIF 34745 - 12/3/9
Boninia glabra Planch
GU no8221 E.H.Wilson
Bonin Islands 1917
Harrisonia abyssinica Oliv.
GU nr3289 G.Taylor
Buganda 1935
Boronia ternata Endl
MJB 1931
Lunasia amara Blanco
Sanko 2955 - 22/5/8
Bouchardatia neurococca ( F.Muell. )
Baill.
Sanko 2949 - 13/5/8
Medicosma subsessilis T.G.Hartley
Sanko 1572 - 29/4/8
Brombya platynema F.Muell.
RDC (Pete Green) - 27/3/8
Myrtopsis novae-caledoniae Engl.
GU Mckee 5041 Ile des Pims
1956
Calodendrum capense Thunb
63114a B Fairchild
Neochamaelea pulverulenta ( Vent. ) GU C Persson 1064
Erdtm.
Citrus wintersii Mabb.
CANB 155240
Nycticalanthus speciosus Ducke
GU Antonelli 499 DUCKE
#3578
Correa lawrenceana var. Grampiana
Paul G. Wilson
MJB 1988
Phebalium squamulosum Vent.
GotBot 1994-V0055 G,
AARHUS bot. Trädg.
Dictyoloma incanescens DC.
GU 4613 Vicosa Ynes
Mexia 1930
Phellodendron amurense Rupr.
GotBot 1946-3851 G,
WEIBULL
Dictyoloma vandellianum A.Juss.
GU 0039124 G.Hatschbach Phellodendron japonicum Maxim.
49448 1985
GotBot 1954-0009 W, Murai
Dinosperma melanophloia
( C.T.White) T.G.Hartley
MJB 1887 - 11/7/7
Pilocarpus pennatifolius Lem.
BELGIUM (XX-0-BR19121462)
Diplolaena drummondii ( Benth. )
Ostenf.
MJB 1956
Pitaviaster haplophyllus ( F.Muell.)
T.G.Hartley
MJB 1964
Diplolaena dampieri Desf.
GU S/N GU w.A. Australia Ravenia spectabilis ( Lindl.) Planch.
Sept 1936
Ex Griseb.
65336A Fairchild
Dutaillyea sessilifoliola Guillaumin
GU Mckee 3848 New
Caledonia 1956
Ruta pinnata L.f.
GU 1407
Empleurum serrulatum Sol.
GU Delagoa bay 21XI
1898 Africa australioccidentali
Sheilanthera pubens I.Williams
GU nr 27899 E.Esterhuysen
Cape Province 1958
Eriostemon australasius Pers
MJB 1869
Skimmia × confusa N.P.Taylor
GotBot 1982-0281 sU
Erythrochiton brasiliensis Nees &
Mart.
GotBot 1962-v2882pG
Skimmia japonica Thunb.
GotBot 1976-3854 pG
Euodia pubifolia T.G.Hartley
cult. ex PIF 25751 31/10/8
Skimmia japonica Thunb.
GotBot 1985-0166 pG
Euodia ridleyi Hochr
91587B Fairchild
Skimmia japonica x intermedia
GotBot Nitz. P-70
Fagara gilletii De Wild.
BELGIUM (XX-0-BR19391845)
Toddalia asiatica Baill.
GU nr 1324 K.Å.Dahlstrand
Transvaal 1963
Flindersia australis R.Br.
RJBGH06-007
Zanthoxylum bonifaziae Cornejo &
Reynel
GU 7141
Flindersia bennettiana F.Muell. Ex
Benth.
ANGB 8301757
Zanthoxylum brachyacanthum
F.Muell.
Sanko 2955
Table 5: The species which have HYB, H&B or MDH marked between the species name and its source
information have been extracted for either of the two genes or both of them.
Cladogram 2 gene Lagrange biogeography. Based on the *BEAST 2 gene species tree. (branch
lengths not to scale):
--------------+
N10+ -----------+
: -N9+
-----+
:
: -N6+
:
: : : --+
N16+
-N8+ -N5+
: :
:
--+
: :
--------+
: :
-----------+
: ---N15+
:
:
-----+
:
---N14+
:
-----+
N38+
: :
----+
: :
--N19+
: :
-N21+
----+
: : -N27+
---------+
: : :
:
---------+
: : :
-N26+
: : :
:
----+
: : :
--N25+
: N37+
----+
:
:
-----------+
N58+
:
:
: :
: N34+
---+
: :
: : :
-N31+
: :
: : -N33+
---+
: :
N36+
:
: :
:
-------+
: :
--------------+
: :
------+
: :
----N41+
: : ----N43+
------+
: : :
-------------+
N62+ N57+ -----------------+
: :
: :
---+
: :
: :
-N47+
: :
N56+
-N49+
---+
: :
: N51+
-------+
: :
N55+ -----------+
N66+ :
:
: : :
:
-------+
: : :
----N54+
: : :
-------+
: : :
-------------+
: : ----------N61+
N70+ :
-------------+
: : :
: : :
--------------+
: : ------------N65+
: :
--------------+
N72+ :
: :
----------------+
: -------------N69+
:
----------------+
-----------------------------------+
[pap] Clymenia_polyandra
[ncd] Oxanthera_neocaledonica
[aus] Eremocitrus_glauca
[aus] Citrus_gracilis
[pap] Microcitrus_papuana
[aus] Microcitrus_australasica
[sin] Poncirus_trifoliata
[ind] Citrus_sinensis
[pap] Citrus_amboiensis
[af1+af2] Citropsis_schweinfurthii
[af1+af2] Citropsis_daweana
[ind+pam+soa+sin] Naringi_crenulata
[pap] Swinglea_glutinosa
[soa+pap] Burkillanthus_malaccensis
[pap] Pleisopermium_latialatum
[soa+sin+pap] Severinia_buxifolia
[ind+soa] Atalantia_monophylla
[soa] Atalantia_citroides
[ind] Atalantia_ceylonica
[ind+pam+soa+sin] Feronia_limonia
[pap] Wenzelia_dolichophylla
[pap] Monanthocitrus_cornuta
[soa+pap] Triphasia_trifolia
[ind] Pamburus_missionis
[af1] Afraegle_paniculata
[af1] Aeglopsis_chevalieri
[af2] Balsamocitrus_dawei
[ind+pam+soa] Aegle_marmelos
[soa] Paramignya_lobata
[ind+soa] Paramignya_scandens
[soa+pap] Merrillia_caloxylon
[ind+pam+soa+sin+aus+pap]
Murraya_paniculata
[soa+pap] Clausena_harmandiana
[ind+pam+soa+sin+pap] Clausena_excavata
[ind+soa+sin+pap] Glycosmis_mauritiana
[soa+pap] Glycosmis_trichanthera
[ind+soa+aus+pap] Micromelum_minutum
At node N5:
[aus|pap]
lnL Rel.Prob At node N6:
-124 1
[aus|aus+pap]
[aus|aus]
lnL Rel.Prob At node N8:
-124.3 0.7178 [aus+pap|aus]
-125.3 0.2666 [aus|aus]
lnL Rel.Prob
-124.3 0.7041
-125.2 0.2882
At node N9:
[ncd|aus+pap]
[ncd|aus]
lnL Rel.Prob At node N10:
-124.6 0.5302 [pap|pap]
-125.2 0.2932 [pap|aus+ncd]
lnL Rel.Prob At node N14:
-124.8 0.4503 [ind|pap]
-125.9 0.1517
lnL Rel.Prob
-124 1
At node N15:
[sin|pap]
[sin|ind+pap]
lnL Rel.Prob At node N16:
-124.6 0.5507 [pap|sin+pap]
-124.8 0.4493 [pap|pap]
[pap|ind+sin+pap]
lnL Rel.Prob At node N19:
-125.4 0.2514 [af2|af2]
-125.5 0.2174 [af1+af2|af1]
-125.5 0.2162
lnL Rel.Prob
-124.4 0.6269
-126.4 0.0921
At node N21:
[af2|ind+soa]
[af2|ind]
[af2|ind+pam+soa]
lnL Rel.Prob At node N25:
-125.5 0.2101 [pap|pap]
-125.9 0.1472
-125.9 0.1425
lnL Rel.Prob At node N26:
-124.1 0.9268 [pap|pap]
lnL Rel.Prob
-124 0.9501
At node N27:
[ind|pap]
[soa|pap]
lnL Rel.Prob At node N31:
-125.1 0.3357 [ind+soa|soa]
-125.9 0.1502
lnL Rel.Prob At node N33:
-124 0.9553
[ind+soa|ind]
[ind|ind]
lnL Rel.Prob
-124.8 0.4368
-124.8 0.4259
At node N34:
[soa|soa]
[soa|ind]
lnL Rel.Prob At node N36:
-125.2 0.2953 [soa|soa]
-125.5 0.2086 [ind|ind]
lnL Rel.Prob At node N37:
-124.9 0.404
[ind+pap|ind]
-125.2 0.2905 [soa+pap|soa]
lnL Rel.Prob
-125.6 0.2015
-125.7 0.1728
At node N38:
[pap|soa+pap]
[pap|pap]
[pap|ind+pap]
lnL Rel.Prob At node N41:
-125.8 0.1558 [pap|pap]
-125.9 0.151
-125.9 0.143
lnL Rel.Prob At node N43:
-124 1
[pap|pap]
[pap|soa+pap]
lnL Rel.Prob
-124.4 0.6825
-125.5 0.2079
At node N47:
[af1|af1]
lnL Rel.Prob At node N49:
-124 1
[af1|af2]
lnL Rel.Prob At node N51:
-124 1
[af2|ind+pam+soa]
[af1+af2|ind]
lnL Rel.Prob
-124.7 0.4626
-125.5 0.2141
At node N54:
[soa|ind+soa]
[soa|soa]
lnL Rel.Prob At node N55:
-124.4 0.6856 [ind|ind]
-125.5 0.2162 [ind+soa|soa]
lnL Rel.Prob At node N56:
-124.4 0.623
[ind|ind]
-126.2 0.1031 [ind|ind+soa]
lnL Rel.Prob
-124.3 0.7415
-125.7 0.1789
At node N57:
[pap|ind]
[soa|soa]
lnL Rel.Prob At node N58:
-124.4 0.6368 [pap|pap]
-126.2 0.1029 [pap|ind+pap]
lnL Rel.Prob At node N61:
-125.7 0.1773 [soa|soa]
-126 0.1273 [soa|
ind+pam+soa+sin+
aus+pap]
[soa|
ind+pam+soa+sin+
pap]
lnL Rel.Prob
-127.1 0.0441
-127.2 0.0405
-127.3 0.0373
At node N62:
[soa|soa]
[pap|pap]
lnL Rel.Prob At node N65:
-125.5 0.2109 [soa|soa]
-125.5 0.2087 [pap|pap]
lnL Rel.Prob At node N66:
-125.6 0.1952 [soa|soa]
-126.6 0.0743 [pap|pap]
lnL Rel.Prob
-124.9 0.3777
-125.5 0.2189
At node N69:
[soa|soa]
[pap|pap]
lnL Rel.Prob At node N70:
-124.9 0.3906 [soa|soa]
-125.9 0.1426 [pap|pap]
lnL Rel.Prob At node N72:
-124.9 0.4157 [soa|soa]
-125.6 0.2047 [pap|pap]
lnL Rel.Prob
-125.7 0.1844
-126.3 0.09571
Table 6: Lagrange relative probabilities for the 2 gene analysis. The table includes the first 1 to 3 values to illustrate
where the node definition is certain and where it is uncertain.
* Split format: [left|right], where 'left' and 'right' are the ranges inherited by each descendant branch (on the printed tree,
'left' is the upper branch, and 'right' the lower branch).
Global ML at root node:
-lnL = 124
dispersal = 0.06009
extinction = 9.832e-12
Cladogram 3 gene Lagrange biogeography. Based on the *BEAST 3 gene species tree.
(branch lengths not to scale):
----------------+
------N4+
:
:
--------+
:
------N3+
:
--------+
:
:
------------------+
:
:
: N19+ ---------------+
N34+ : : :
: : : :
---------+
: : N18+ N13+
: :
: : : ------+
: :
: : N12+
: :
: :
: ---+
: :
N17+
N11+
: :
:
---+
: :
:
N33+
:
------+
:
---N16+
:
------+
:
:
------+
:
--N22+
:
:
------+
:
--N26+
:
:
:
------+
:
:
--N25+
--N32+
------+
:
:
-----------+
--N31+
:
------+
--N30+
------+
[soa+pap] Clausena_harmandiana
[ind+pam+soa+sin+aus+pap] Murraya_paniculata
[soa+pap] Merrillia_caloxylon
[pap] Swinglea_glutinosa
[ind+pam+soa+sin] Naringi_crenulata
[sin] Poncirus_trifoliata
[ind] Citrus_sinensis
[aus] Citrus_gracilis
[aus] Microcitrus_australasica
[ind+soa] Atalantia_monophylla
[ind] Atalantia_ceylonica
[af1] Afraegle_paniculata
[af1] Aeglopsis_chevalieri
[af2] Balsamocitrus_dawei
[ind+pam+soa] Aegle_marmelos
[pap] Monanthocitrus_cornuta
[soa] Paramignya_lobata
[ind] Pamburus_missionis
At node N3:
[pap|pap]
[soa|soa]
[ind+pam+soa+sin+
aus+pap|soa]
[ind+pam+soa+sin+
aus+pap|pap]
lnL Rel.Prob At node N4:
-64.17 0.0460 [pap|pap]
-64.34 0.0388 [soa|soa]
-64.44 0.0348
lnL Rel.Prob At node N11:
-62.5 0.243
[aus|aus]
-62.65 0.2101 [aus+pap|aus]
lnL Rel.Prob
-61.26 0.846
-63.73 0.071
At node N12:
[ind+pap|aus]
[ind|aus+pap]
lnL Rel.Prob At node N13:
-61.91 0.4384 [sin|ind+aus+pap]
-62.32 0.2925 [sin|ind+pap]
lnL Rel.Prob At node N16:
-61.77 0.5057 [ind|ind]
-62.64 0.2124 [ind+soa|ind]
lnL Rel.Prob
-61.26 0.8434
-63.49 0.0909
At node N17:
[ind|ind]
[ind+pap|ind]
lnL Rel.Prob At node N18:
-62.05 0.3837 [ind|ind]
-62.91 0.1619 [ind|ind+pap]
lnL Rel.Prob At node N19:
-62.34 0.2856 [ind|ind]
-63.17 0.1247 [pap|ind+pap]
[pap|ind]
[pap|pap]
lnL Rel.Prob
-63
0.1472
-63.16 0.1253
-63.26 0.1137
-63.33 0.1067
At node N22:
[af1|af1]
lnL Rel.Prob At node N25:
-61.09 0.9974 [af2|ind+pam+soa]
[af2|ind+pam]
lnL Rel.Prob At node N26:
-61.24 0.8547 [af1|
-64.14 0.0473 af2+ind+pam+soa]
[af1|af2+ind+pam]
lnL Rel.Prob
-61.72 0.532
lnL Rel.Prob
-61.95 0.4202
-62.99 0.1494
-64.51 0.0327
At node N30:
[ind|ind]
[soa|ind]
lnL Rel.Prob At node N31:
-62.22 0.3209 [ind|ind]
-62.5 0.2435 [soa|soa]
lnL Rel.Prob At node N32:
-62 0.4031
[ind|ind]
-62.54 0.2333 [soa|soa]
At node N33:
[ind|ind]
[ind+pap|ind]
lnL Rel.Prob At node N34:
-62.58 0.225
[pap|ind+pap]
-63.97 0.0559 [pap|pap]
[soa|soa]
lnL Rel.Prob
-64.51 0.0325
-64.85 0.0231
-64.96 0.0208
-63.27 0.113
Table 7: Lagrange relative probability table for the 3 gene analysis. The table includes the first 1 to 4 values to
illustrate where the division is certain and where it is uncertain.
* Split format: [left|right], where 'left' and 'right' are the ranges inherited by each descendant branch (on the printed tree,
'left' is the upper branch, and 'right' the lower branch).
Global ML at root node:
-lnL = 61.09
dispersal = 0.03715
extinction = 0.03039
[CTAB mixing
To make 100 ml of the CTAB buffer mentioned in the above protocol (3), dissolve 8.85 g of TRISHCl with 5.3 g of TRIS-base with MQ (milli-q water) and fill until you reach 45 ml. In a different
beaker, mix 1.82 g of CTAB with MQ and once dissolved, add until you have 45 ml total volume.
This can be quickly dissolved with either a heating stirrer or with the help of a microwave. 18.6 g of
EDTA is needed for this buffer. Take half of this amount and mix it with the TRIS solution. When it
is dissolved, add some of the CTAB solution and some more of the EDTA buffer until everything is
in solution. Each M of NaCl needed weighs 5.84 g. Add the NaCl one M at a time to let it dissolve.
NaCl releases gas in the liquid as it dissolves. Therefore, as low viscosity as possible is wanted.
Warm the solution to 65 C while stirring in order to dissolve the NaCl well. Continue until desired
NaCl molarity has been reached.]
Acknowledgements
I would like to thank Bernard Pfeil for excellent supervising of my thesis work. I would also like to
thank Mats Töpel, Stephan Nylinder and Vivian Alden as well the rest of the Molecular Systematics
Research Group, for introducing me to various parts of the land of Systematica Botanica. I am
grateful for the contributions made by Gothenburg Botanical Garden; Australian National Botanic
Gardens; Fairchild Tropical Botanic Garden; the National Botanic Garden of Belgium; Claes
Persson and Alex Antonelli (University of Gothenburg and Gothenburg Botanical Garden) who
graciously provided me with leaf samples. I also thank Cathy Miller (CSIRO, Canberra) and Mike
Bayly (University of Melbourne) for providing some extracted DNA samples. The following
herbaria also provided material for study and in some cases DNA extraction: GB, MO – my thanks
to them as well. Part of this work was carried out by using the resources of the Computational
Biology Service Unit from Cornell University which is partially funded by Microsoft Corporation.
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