RESEARCH ARTICLE INVITED SPECIAL ARTICLE For the Special Issue: Using and Navigating the Plant Tree of Life Organellar phylogenomics inform systematics in the green algal family Hydrodictyaceae (Chlorophyceae) and provide clues to the complex evolutionary history of plastid genomes in the green algal tree of life Hilary A. McManus1,5, Karolina Fučíková2, Paul O. Lewis3, Louise A. Lewis3, and Kenneth G. Karol4 Manuscript received 5 July 2017; revision accepted 19 December PREMISE OF THE STUDY: Phylogenomic analyses across the green algae are resolving 2017. relationships at the class, order, and family levels and highlighting dynamic patterns of 1 Department of Biological and Environmental Sciences, Le evolution in organellar genomes. Here we present a within- family phylogenomic study to Moyne College, 1419 Salt Springs Road, Syracuse, New York resolve genera and species relationships in the family Hydrodictyaceae (Chlorophyceae), for 13066, USA which poor resolution in previous phylogenetic studies, along with divergent morphological 2 Department of Natural Sciences, Assumption College, 500 Salisbury Street, Worcester, Massachusetts 01609, USA traits, have precluded taxonomic revisions. 3 Department of Ecology and Evolutionary Biology, University of METHODS: Complete plastome sequences and mitochondrial protein- coding gene sequences Connecticut, Storrs, Connecticut 06269, USA were acquired from representatives of the Hydrodictyaceae using next- generation 4 Lewis B. and Dorothy Cullman Program for Molecular sequencing methods. Plastomes were characterized, and gene order and content were Systematics, The New York Botanical Garden, 2900 Southern Boulevard, Bronx, New York 10458, USA compared with plastomes spanning the Sphaeropleales. Single- gene and concatenated- gene 5 Author for correspondence (e-mail: [email protected]) phylogenetic analyses of plastid and mitochondrial genes were performed. Citation: McManus, H. A., K. Fučíková, P. O. Lewis, L. A. Lewis, and KEY RESULTS: The Hydrodictyaceae contain the largest sphaeroplealean plastomes thus K. G. Karol. 2018. Organellar phylogenomics inform systematics in far fully sequenced. Conservation of plastome gene order within Hydrodictyaceae the green algal family Hydrodictyaceae (Chlorophyceae) and provide is striking compared with more dynamic patterns revealed across Sphaeropleales. clues to the complex evolutionary history of plastid genomes in the green algal tree of life. American Journal of Botany 105(3): 315–329. Phylogenetic analyses resolve Hydrodictyon sister to a monophyletic Pediastrum, though doi:10.1002/ajb2.1066 the morphologically distinct P. angulosum and P. duplex continue to be polyphyletic. Analyses of plastid data supported the neochloridacean genus Chlorotetraëdron as sister to Hydrodictyaceae, while conflicting signal was found in the mitochondrial data. CONCLUSIONS: A phylogenomic approach resolved within- family relationships not obtainable with previous phylogenetic analyses. Denser taxon sampling across Sphaeropleales is necessary to capture patterns in plastome evolution, and further taxa and studies are needed to fully resolve the sister lineage to Hydrodictyaceae and polyphyly of Pediastrum angulosum and P. duplex. KEY WORDS Chlorophyta; chloroplast; Hydrodictyon; monophyly; organelle; Pediastrum; phylogeny; plastome; Sphaeropleales. American Journal of Botany 105(3): 315–329, 2018; http://www.wileyonlinelibrary.com/journal/AJB © 2018 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. • 315 316 • American Journal of Botany Studies using phylogenomic data, particularly complete organellar and Pokorný, 2002; Lezine et al., 2005; Milecka and Szeroczýnska, genomes, are becoming standard, and the field of plant evolution 2005; Weckström et al., 2010). has seen an increase of such studies focusing on family-level ques- Historically, four genera have been recognized in the tions in particular (e.g., Henriquez et al., 2014; Guo et al., 2017). Hydrodictyaceae: Euastropsis Lagerheim 1895, Hydrodictyon Although organellar genome data have contributed valuable in- Roth 1797, Pediastrum Meyen 1829, and Sorastrum Kützing 1845. formation for higher- level systematics in phycology (e.g., Brouard However, several molecular phylogenetic studies, using two or three et al., 2010), until recently most studies have focused on sampling targeted DNA sequence regions, have consistently yielded Pediastrum one or a few representatives per major lineage for organellar ge- as paraphyletic. As a result, five additional genera have been recog- nome sequencing. Phylogenomic studies in green algae have re- nized: Lacunastrum H.McManus 2011, Monactinus Corda 1839, vealed numerous patterns and processes of evolution typically not Parapediastrum E.Hegewald 2005, Pseudopediastrum E.Hegewald reported in land plant plastomes. For example, extensive size vari- 2005, and Stauridium Corda 1839 (Buchheim et al., 2005; McManus ation within and among orders, as well as highly rearranged gene and Lewis, 2005, 2011; McManus et al., 2011). Though significant orders and gene gain/loss, are characteristic of the green algal lin- progress has been made in Hydrodictyaceae phylogeny and classifi- eages so far examined in this respect (Smith et al., 2013; Turmel cation, several unresolved questions remain. For example, the place- et al., 2015; Fučíková et al., 2016a, 2016b). The green algal order ment of Hydrodictyon is problematic, being resolved as either sister Sphaeropleales has recently received more attention with a broad to a monophyletic Pediastrum (Buchheim et al., 2005; McManus and sampling of representatives and increased number of molecular Lewis, 2005; Jena et al., 2014; Lenarczyk and McManus, 2016), or markers and whole organellar genomes analyzed (Farwagi et al., embedded within it rendering Pediastrum paraphyletic (Buchheim 2015; Lemieux et al., 2015; Fučíková et al., 2014b, 2014c, 2016a, et al., 2005; McManus and Lewis, 2011). Additionally, Pediastrum 2016b; McManus et al., 2017). A study of nine mitochondrial ge- contains two species (P. angulosum Ehrenberg ex Meneghini 1840 nomes within the order demonstrated conserved gene content but and P. duplex Meyen 1829), neither of which has been strongly sup- variability in intron content and genome size, and gene order analy- ported as monophyletic. A subset of P. duplex (“Group I”; McManus ses indicated potential to help resolve phylogenetic relationships at and Lewis, 2011) has been generally recovered, while the remainder the order and family levels (Fučíková et al., 2014b, 2014c; Farwagi of P. duplex has been recovered with P. angulosum as a polyphyletic et al., 2015). Characterization of the plastomes found extensive var- cluster (“Group II”; McManus and Lewis, 2011). The high genetic iation in overall size and inverted repeats (IR), and phylogenomic similarity and low nodal support, combined with considerable mor- analyses revealed support for different topologies depending on phological differences between Hydrodictyon, P. angulosum, and P. whether nucleotide or inferred amino acid sequences were used duplex, precluded taxonomic revisions. (Fučíková et al., 2016a). Clearly, we are still in the process of uncov- Within Hydrodictyaceae, mitochondrial gene order analyses ering the complexities of organellar genome evolution in green al- of four representatives indicated potential for resolving phyloge- gae. Denser taxon sampling within orders and families is the logical netic relationships (Farwagi et al., 2015), and a comparison of next step, which will allow addressing taxonomic issues associated two fully sequenced plastomes, one from Hydrodictyon reticula- with limited or convergent morphology that is a common problem tum (Linnaeus) Bory 1824 and the other from Pediastrum duplex, in microscopic green algae, and may uncover novel processes of or- showed that the two taxa shared gene order and plastome struc- ganellar genome evolution that help explain patterns seen at more ture, were more similar to each other than to other members of the inclusive taxonomic levels. Sphaeropleales and represented the largest plastomes known in the The Hydrodictyaceae (Sphaeropleales, Chlorophyta) is a remark- order (McManus et al., 2017). A phylogenetic analysis of plastome able group of green algae not only because of unique evolutionary gene order within Sphaeropleales supported the sister relationship trends discovered in their chloroplast and mitochondrial genomes, of Hydrodictyon and Pediastrum, but taxon sampling limited tests but also because of their evolving classification. They are found in of Pediastrum monophyly. freshwater and brackish systems worldwide and include ecologi- This study presents new plastomes from representatives cally important planktonic forms as well as nuisance bloomers. The of the Hydrodictyaceae, including Lacunastrum, Pediastrum, family is defined by their gametic flagellar apparatus ultrastruc- Pseudopediastrum, and Stauridium, and describes plastome archi- ture, similar asexual and sexual life cycle traits (Appendix S1, see tecture and gene order in the context of Sphaeropleales. We con- Supplemental Data with this article), and the formation of colonies ducted phylogenomic