PROBING THE MONOPHYLY OF THE SPHAEROPLEALES (CHLOROPHYCEAE) USING DATA FROM FIVE GENES 1 Nicholas P. Tippery, 2 Karolina Fucˇı´kova´, Paul O. Lewis, and Louise A. Lewis 3 Department of Ecology and Evolutionary Biology, University of Connecticut, 75 North Eagleville Road, Storrs, Connecticut 06269, USA Molecular phylogenetic analyses have had a major impact on the classification of the green algal class Chlorophyceae, corroborating some previous evolutionary hypotheses, but primarily promoting new interpretations of morphological evolution. One set of morphological traits that feature prominently in green algal systematics is the absolute orientation of the flagellar apparatus in motile cells, which correlates strongly with taxonomic classes and orders. The order Sphaeropleales includes diverse green algae sharing the directly opposite (DO) flagellar apparatus orientation of their biflagellate motile cells. However, algae across sphaeroplealean families differ in specific components of the DO flagellar apparatus, and molecular phylogenetic studies often have failed to provide strong support for the monophyly of the order. To test the monophyly of Sphaeropleales and of taxa with the DO flagellar apparatus, we conducted a molecular phylogenetic study of 16 accessions representing all known families and diverse affiliated lineages within the order, with data from four plastid genes (psaA, psaB, psbC, rbcL) and one nuclear ribosomal gene (18S). Although single-gene analyses varied in topology and support values, analysis of combined data strongly supported a monophyletic Sphaeropleales. Our results also corroborated previous phylogenetic hypotheses that were based on chloroplast genome data from relatively few taxa. Specifically, our data resolved Volvocales, algae possessing predominantly biflagellate motile cells with clockwise (CW) flagellar orientation, as the monophyletic sister lineage to Sphaeropleales, and an alliance of Chaetopeltidales, Chaetophorales, and Oedogoniales, orders having multiflagellate motile cells with distinct flagellar orientations involving the DO and CW forms. Key index words: Bayesian analysis; coccoid green algae; flagellar apparatus; phylogenetic information; polytomy analysis Abbreviations: 18S, nuclear small subunit ribosomal DNA; CW, clockwise; CCW, counterclockwise; DO, directly opposite; KL, Kullback-Leibler; psaA, photo- system I chlorophyll a-apoprotein A1; psaB, photosys- tem I chlorophyll a-apoprotein A2; psbC, photosystem II chlorophyll a-apoprotein CP43; rbcL, ribulose bis- phosphate carboxylase/oxygenase large subunit Green algae in the class Chlorophyceae comprise morphologically diverse lineages that grow in fresh- water or terrestrial environments worldwide. Prior to molecular phylogenetic analyses, these algae were extraordinarily challenging to classify reliably, owing to their often reduced forms and the particular life stages or high magnifications at which taxonomi- cally informative characters must be observed. Within Chlorophyceae, algae in the order Sphaero- pleales have been grouped by molecular data and by the shared ultrastructural feature of biflagellate motile cells with directly opposite (DO) flagellar apparatus absolute orientation (referred to herein as “flagellar orientation”; Mattox and Stewart 1984, Deason et al. 1991, Lewis and McCourt 2004). Among chlorophycean orders, the Volvocales (con- sidered here to include Chlamydomonadales) are a lineage having bi- or quadriflagellate motile cells with clockwise (CW) flagellar orientation and are most closely related to Sphaeropleales, whereas Chaetopeltidales (quadriflagellate DO + DO), Chae- tophorales (quadriflagellate DO + CW), and Oedog- oniales (motile cells with an anterior ring of flagella) are more distantly related (Lewis and McCourt 2004, Pro ¨schold and Leliaert 2007, Leliaert et al. 2012). Although analyses of molecular data generally have supported the monophyly of orders and the relation- ships among them, a comprehensive phylogenetic assessment requires a more thorough inclusion of taxa and genes than has been achieved previously. Sphaeropleales includes diverse and ecologically important taxa from both freshwater and terrestrial habitats, such as the morphologically distinct aquatics Ankistrodesmus Corda, Desmodesmus (R. Cho- dat) S. S. An, T. Friedl & E. Hegewald, Pediastrum Meyen, and Scenedesmus Meyen, the bloom-forming Hydrodictyon Roth, and the terrestrial Bracteacoccus Tereg. Several genera in this order (e.g., Ankis- trodesmus, Desmodesmus, Pediastrum, Scenedesmus) are used routinely as ecological indicator species (e.g., Magdaleno et al. 1997, McCormick and Cairns 1997, Koma ´rek and Jankovska ´ 2001, Weckstro ¨m 1 Received 31 August 2011. Accepted 29 April 2012. 2 Present address: Department of Biological Sciences, University of Wisconsin—Whitewater, 800 West Main Street, Whitewater, Wiscon- sin 53190, USA. 3 Author for correspondence: e-mail louise.lewis@uconn.edu. J. Phycol. 48, 1482–1493 (2012) © 2012 Phycological Society of America DOI: 10.1111/jpy.12003 1482