The Characeae is a family of macrophytic green algae closely related to land plants (Lewis & McCourt, 2004). The group is globally distributed (except for Antarctica) and can be readily found in many freshwater lakes (Dodds, 2002). Even though these algae can dramatically influence nutrient loads and water quality where they occur (Kufel & Kufel, 2002; Kufel & Ozimek, 1994), a misleading and incomplete understanding of the species within this group makes well informed management strategies for these organisms and the ecosystems in which they reside difficult.
Figure 1-A chloroplast four gene RaxML phylogeny using the genes rbcL, psbC, atpB, ndhD. Colored boxes indicate previously recognized species within the Willdenowia. Red boxes indicate dioecious taxa, blue indicate sejoined taxa, and yellow indicate conjoined taxa. Chromosome counts are included where known. Bootstrap support (BS) values greater than 50 are included, BS values of 100 are represented by asterices.
Figure 2-Part of the nuclear encoded ribosomal operon is shown. The 5.8S and 26S ribosomal genes are shown in orange and are separated by ITS2 in blue. Primer locations (red arrows) used for this study were located at the 5 ends of the 5.8S and 26S segments.
Figure 3-PCR products run on a 1% agarose gel. A = Klebsormidium, B = KGK4770 (C. foliolosa), C = KGK4839 (C. rusbyana), D = KGK2150 (C. zeylanica), E = strain KGK0008 (C. foliolosa), F = KGK0192 (C. zeylanica), G = KGK0196 (C. brittonii), H = KGK0466 (C. hydropitys).
Figure 4-comparison of the nuclear ITS2 (left) and chloroplast four gene (right) phylogenies. The major species-clades from the chloroplast phylogeny and their corresponding taxa in the ITS2 nuclear phylogeny are boxed with corresponding colors. Though the C. haitensis group changes position with C. rusbyana in the two phylogenies, this does not appear to be a strongly supported rearrangement in either case. Published sequences are used to fill out the ITS2 phylogeny (Hall et al., 2010). Notably, C. rusbyana, a cloned and resequenced individual represented as KGK4839-2 and KGK4839-1 have 30 SNPs with respect to one another, possibly due to multiple alleles for this locus. This causes them to appear paraphyletic in the nuclear ITS2 phylogeny.
Chara subsection Willdenowia (Charophyta) is a monophyletic group within the Characeae, and has been considered one cosmopolitan species - Chara zeylanica - since the most recent revision of the group in 1965. Four gene chloroplast DNA sequences and other data strongly contradict the single-species hypothesis. However, a phylogenetically informative nuclear marker would be of value for this group. Sequencing data indicate that chloroplast DNA is maternally inherited, and so could represent an alternative phylotgenetic signal to a nuclear marker, particularly if hybridization between clades is taking place. An altered topology between a Willdenowia nuclear phylogeny and chloroplast phylogeny could indicate hybridizations are taking place and would better inform species circumscription for the group. The nuclear Internal Transcribed Spacer 2 (ITS2) marker has been suggested as a universal barcode for species identification and phylogenetics in plants, animals, and fungi. ITS2 was sequenced for members of the Willdenowia and a phylogeny comprised of these and published sequences was compared with the chloroplast phylogeny.
Thank you to Catharina Grubaugh and Anthony Evans for tremendous patience, support, and helpful suggestions throughout the project. Thank you Dr. Berish Rubin for guidance and insightful comments. This work could not have been carried out without the generous contribution of DNA and live specimens from the Karol Lab and the New York Botanical Garden.
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