Alignment of all phased ITS2 individuals

Sample names are in the following format: indentifier_species_(collection site). The letters a and b suffixed to the identifier denote the alleles for the same individual. Homozygotes are entered twice. Individuals were phased using a combination of the program PHASE 2.1 as implemented in DNASP v5 and Indelligent v1.2 (available from: imperialis.inhs.illinois.edu/dmitriev/indel.asp‎)

A New Classification of <i>Ficus</i> Subsection <i>Urostigma</i> (Moraceae) Based on Four Nuclear DNA Markers (ITS, ETS, G3pdh, and ncpGS), Morphology and Leaf Anatomy

<div><p><i>Ficus</i> subsection <i>Urostigma</i> as currently circumscribed contains 27 species, distributed in Africa, Asia, Australia and the Pacific, and is of key importance to understand the origin and evolution of <i>Ficus</i> and the fig-wasp mutualism. The species of subsection <i>Urostigma</i> are very variable in morphological characters and exhibit a wide range of often partly overlapping distributions, which makes identification often difficult. The systematic classification within and between this subsection and others is problematic, e.g., it is still unclear where to classify <i>F</i>. <i>amplissima</i> and <i>F</i>. <i>rumphii</i>. To clarify the circumscription of subsection <i>Urostigma</i>, a phylogenetic reconstruction based on four nuclear DNA markers (ITS, ETS, G3pdh, and ncpGS) combined with morphology and leaf anatomy is conducted. The phylogenetic tree based on the combined datasets shows that <i>F</i>. <i>madagascariensis</i>, a Madagascan species, is sister to the remainder of subsect. <i>Urostigma</i>. <i>Ficus amplissima</i> and <i>F</i>. <i>rumphii</i>, formerly constituting sect. <i>Leucogyne</i>, appear to be imbedded in subsect. <i>Conosycea</i>. The result of the phylogenetic analysis necessitates nomenclatural adjustments. A new classification of <i>Ficus</i> subsection <i>Urostigma</i> is presented along with the morphological and leaf anatomical apomorphies typical for the clades. Two new species are described ─ one in subsect. <i>Urostigma</i>, the other in <i>Conosycea</i>. One variety is raised to species level.</p></div

Genbank accession numbers

A table of genbank accession numbers for all specimens collected during this study. Additional columns are included indicating the species and sampling site for each specimen

Total evidence MCC tree fromBayesian analysis of four DNA markers, morphology and leaf anatomy.

<p>Posterior probabilities (PP) above and bootstrap supports (BS) below the branches.</p

Sequences of primers used in this study.

<p>Notes: The primer combinations ITS_5F plus ITS 4R and ITS17SE plus ITS26SE were used interchangingly with about equal success corresponding to standard protocols at C and CNRS. The combination of the <i>Ficus</i> specific internal primer ETS_Fig1_F plus 18S_ETS was only used for amplification of 13 accessions across the subsection, which could not be amplified with the standard primers.</p><p>Sequences of primers used in this study.</p

Evolution of some selected morphological and leaf anatomical characters optimized onto the phylogeny tree (Fig 2) using Mesquite v.2.7.5 with parsimony.

<p>A: Intermittent growth (character 3), B: Deciduous leaves (character 7), C: Staminate flowers (character 40), and D: Enlarged lithocysts (character 47).</p

Parsimony distribution of the morphological characters and their states on the total evidence MCC Bayesian tree (Fig 2).

<p>● = unique apomorphy; ○ = parallelism; ✕ = reversal; ⨂ = parallel reversal.</p

Maximum Clade Credibility (MCC) tree from Bayesian analysis of four combined DNA markers (ITS, ETS, G3pdh, and ncpGS) with posterior probabilities (PP) above and bootstrap supports (BS) below the branches.

<p>Maximum Clade Credibility (MCC) tree from Bayesian analysis of four combined DNA markers (ITS, ETS, G3pdh, and ncpGS) with posterior probabilities (PP) above and bootstrap supports (BS) below the branches.</p

<i>Ficus pseudocaulocarpa</i> Chantaras.

<p>A: Twig with leaves and figs. B: Fig. C: Fig in longitudinal section. D: Staminate flower. E and F: Pistillate flowers.-Drawing: Pajaree Inthachup, 2014.</p

<i>Ficus pubipetiola</i> Chantaras.

<p>A: Twig with leaves and figs. B: Fig. C: Fig in longitudinal section. D: Staminate flower. E: Anther. F and G: Pistillate flowers.-Drawing: Pajaree Inthachup, 2014.</p