• coccoid;
  • cryptic genera;
  • psaB;
  • psbC;
  • rbcL;
  • rDNA ;
  • taxonomic revision;
  • tufA


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Supporting Information

Best known for aquatic colonial algae such as Hydrodictyon, Pediastrum, or Scenedesmus, the order Sphaeropleales also contains numerous coccoid taxa from aquatic and terrestrial habitats. Recent findings indicate that coccoid lineages in this order are very diverse genetically and may be the prevalent form, although their diversity is often hidden morphologically. This study characterizes coccoid algae recently discovered from desert soil crusts that share morphological and ecological features with the genera Bracteacoccus, Pseudomuriella, and Chromochloris. Analyses of a multi-gene data set that includes members from all sphaeroplealean families are used to examine the monophyly of these morphologically similar taxa, which are shown instead to be phylogenetically distinct and very divergent. We propose new generic names for these lineages: Bracteamorpha, Rotundella, and Tumidella. In addition, we propose an updated family-level taxonomy within Sphaeropleales that includes ten new families of coccoid algae to accommodate the newly presented genera and many incertae sedis taxa in the order: Bracteamorphaceae, Chromochloridaceae, Dictyococcaceae, Dictyochloridaceae, Mychonastaceae, Pseudomuriellaceae, Rotundellaceae, Schizochlamydaceae, Schroederiaceae, and Tumidellaceae.

List of Abbreviations

nuclear small subunit rDNA


28S nuclear large subunit rDNA


5.8S nuclear large subunit rDNA


PSI chlorophyll a-apoprotein A2


PSII chlorophyll a-apoprotein CP43


ribulose bisphosphate carboxylase/oxygenase large subunit


elongation factor TU

Vegetatively unicellular, spherical, nonmotile (coccoid) green algae are found commonly in aquatic and terrestrial habitats worldwide, including extreme environments such as postmining dumps, polar arid soils, and deserts (e.g., Broady 1986, Flechtner et al. 1998, Patova and Dorokhova 2008). Historically, many coccoid genera were assigned to the order Chlorococcales based on gross morphological similarities, but this order is now understood as a polyphyletic assemblage of taxa distributed into the classes Chlorophyceae, Trebouxiophyceae, and Ulvophyceae (reviewed in Lewis and McCourt 2004). Remarkable diversity of such morphologically simple algae has been unearthed by recent biodiversity and systematics studies that used molecular phylogenetics (e.g., Lewis and Flechtner 2004, Lewis and Lewis 2005, Fučíková et al. 2011b, 2013, Flechtner et al. 2013).

Within the chlorophycean order Sphaeropleales, several genera possess the coccoid morphology, as well as multiple chloroplasts and nuclei: Bracteacoccus, Chromochloris, Dictyococcus, Follicularia, Planktosphaeria, and Pseudomuriella. All of the above-named genera reproduce asexually via biflagellate naked zoospores. Past phylogenetic investigations illustrated weak support for the monophyly of these taxa, and it was previously hypothesized that this morphology and life history might be monophyletic within Sphaeropleales (Fučíková and Lewis 2012, Fučíková et al. 2013). In this study, we examined the hypothesis of monophyletic Bracteacoccus-like algae by characterizing three new Bracteacoccus-like lineages, and using phylogenetic analyses of the nuclear rDNA genes (28S, 5.8S, and 18S) and four protein-coding chloroplast genes (psaB, psbC, rbcL, and tufA) in the context of even taxon sampling across Sphaeropleales. In addition to strains obtained from public culture collections, four strains isolated from desert soil crusts from North America (Carlsbad Caverns Nat. Park, NM, USA; Joshua Tree Nat. Park, CA, USA; Zion Nat. Park, UT, USA) and Africa (Namibia) were examined.

The established families within the order Sphaeropleales are Hydrodictyaceae, Neochloridaceae, Radiococcaceae, Scenedesmaceae, Selenastraceae (syn. Ankistrodesmaceae), Sphaeropleaceae, and the recently erected Bracteacoccaceae. In addition, many genera are regarded as incertae sedis within Sphaeropleales, i.e., are without a family-level affiliation. Many of these are unicellular algae morphologically similar to one another, but molecular phylogenetic analyses demonstrate them to be deeply diverging lineages (Tippery et al. 2012). Analysis of the multilocus data set, the most comprehensive for Sphaeropleales to date, also allowed us to address family-level taxonomy within the order. Even though relationships among most families were still impossible to resolve with confidence, we were able to make taxonomic decisions based on phylogenetic distinctness of well-supported clades. On the basis of our results, we assign some of the incertae sedis genera in Sphaeropleales to existing families and propose ten new families based on phylogenetic evidence.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Supporting Information

Algal cultures were obtained from either the Culture Collection of Algae at the University of Göttingen, Germany (SAG; or the Culture Collection of Algae at the University of Texas at Austin, USA (UTEX;, as well as from newly collected material (Table 1 and Table S1 in the Supporting Information). Soil was collected previously as part of the Biotic Crust Project (BCP,, and the strains were isolated into unialgal cultures by L. Lewis, V. Flechtner, and N. Pietrasiak (personal communication) following the methods described in Flechtner et al. (1998). Strain BCP-CC1VF5A was deposited at the UTEX collection as UTEX B2977 and strain BCP-WJT54VFNP11 was deposited as UTEX B2979. Cultures were maintained on agar slants containing Bold's Basal Medium (BBM, Bold 1949, Bischoff and Bold 1963) and BBM enriched with soil water extract, under 16:8 light:dark cycle at 18°C and 70 μmol photons · m−2 · s−1. Cell morphology across life cycle stages was examined using an Olympus BX60 light microscope with Nomarski DIC optics (Olympus Imaging America Inc., Center Valley, PA, USA). Zoospore and gamete induction was carried out by flooding and light starvation (Fučíková et al. 2013).

Table 1. List of algal strains used in this study with the corresponding GenBank accession numbers for the seven genes of interest. In Scenedesmus obliquus, the 18S sequence of the strain UTEX 1450 was used instead of the closely related UTEX 393 due to availability from GenBank. Sequences obtained for this study are highlighted in boldface font and n/a indicates that sequencing was unsuccessful
Ankistrodesmus falcatus UTEX 101 JN630515 KC145448 KC145459 JN630538 JN630559 JQ394814 KC145525
Ankyra judai SAG 17.84 U73469 AF183448 KC145467 KC145484 KC145506 EF113408 KC145526
Botryosphaerella sudetica UTEX 2629 AJ581914 KC145453 AJ581914 KC145488 KC145503 KC145508 n/a
Bracteacoccus aerius UTEX 1250 U63101 KC145456 JQ281839 JN630540 JN630561 GQ985398 JQ281879
Bracteacoccus minor UTEX 66 U63097 AF183452 JF717398 JN630541 JN630562 GQ985399 JQ281889
Bracteamorpha trainorii UTEX B2977 JQ259955 KC145441 JQ281874 KC145478 KC145494 JQ259909 JQ281897
Characiopodium hindakii UTEX 2098 M63000 AF183466 n/a JN630543 JN630564 EF113418 n/a
Chlorotetraedron incus SAG 43.81 AF288363 KC145442 n/a KC145479 KC145495 KC145512 KC145520
Chromochloris zofingiensis UTEX 56 HQ902933 KC145443 HQ902927 JN630545 JN630566 HQ902939 HQ902930
Dictyochloris fragrans UTEX 127 AF367861 AY206711 KC145463 KC145480 KC145500 KC145513 KC145521
Dictyococcus varians UTEX LB 62 GQ985408 KC145452 KC145469 KC145487 KC145502 GQ985404 JQ281896
Follicularia botryoides UTEX LB 951 KC145433 KC145449 KC145468 KC145485 KC145501 JQ259910 KC145527
Follicularia texensis UTEX 1241 JN630516 KC145444 KC145473 JN630553 JN630574 JQ259912 JQ281899
Hariotina reticulata UTEX 1365 AH012395 KC145450 GQ375101 JN630546 JN630567 JQ394815 KC145528
Kirchneriella aperta SAG 2004 AJ271859 KC145445 KC145464 KC145481 KC145497 KC145514 KC145522
Mychonastes homosphaera CAUP H6502 GQ477056 KC145446 GU799581 KC145482 KC145498 KC145515 KC145523
Mychonastes jurisii SAG 37.98 AF106074 KC145447 GQ477038 EU380566 KC145499 EU380520 n/a
Neochloris aquatica UTEX 138 M62861 AF277653 AY577764 JN630548 JN630569 EF113456 KC145529
Ourococcus multisporus UTEX 1240 AF277648 AF277655 KC145470 JN630550 JN630571 EF113460 KC145530
Parapediastrum biradiatum UTEX 37 AY663034 AY779881 KC145471 KC145486 KC145507 EF078303 n/a
Pediastrum duplex UTEX LB 1364 AY779859 AF183479 AY577734 JN630551 JN630572 EF113461 KC145531
Planktosphaeria gelatinosa SAG 262-1b AY044648 KC145451 JQ281876 JN630532 JN630573 HM852435 JQ281898
Pseudomuriella engadinensis UTEX 58 HM852442 KC145454 HQ292730 KC145489 KC145504 HM770959 HQ292755
Pseudomuriella schumacherensis SAG 2137 HQ292768 KC145457 HQ292724 JN630555 JN630576 HQ292737 HQ292746
Radiococcus polycoccus SAG 217-1c AF388378 KC145455 JQ281875 KC145490 KC145505 HM852437 JQ281900
Rotundella rotunda UTEX B2979 KC145434 KC145437 KC145460 KC145474 KC145491 KC145509 KC145517
Rotundella sp.BCP-ZNP1VF31 KC145435 KC145439 KC145461 KC145476 n/a KC145510 KC145518
Scenedesmus obliquus UTEX 393 AJ249515 KC145458 AJ249505 NC_008101 NC_008101 NC_008101 DQ396875
Scenedesmus rotundus BCP-SEV3VF49 AF513373 KC145438 AY510465 KC145475 KC145492 HQ246350 HQ246371
Schizochlamys gelatinosa SAG 66.94 AY781662 AY779895 KC145466 KC145483 KC145496 KC145516 KC145524
Schroederia setigera UTEX LB2454 AF277650 AF277657 KC145465 JN630557 JN630578 EF113470 KC145532
Sphaeroplea robusta UTEX 2350 U73472 AF183484 KC145472 JN630558 JN630579 EF113472 KC145533
Tumidella tumida SAG 2265 KC145436 KC145440 KC145462 KC145477 KC145493 KC145511 KC145519

DNA was isolated using the PowerPlant DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA). Primers and PCR conditions from Shoup and Lewis (2003) were used for the 18S and 28S genes. Primers and conditions used for PCR amplification and cycle sequencing of rbcL are listed in McManus and Lewis (2011), for psaB and psbC in Tippery et al. (2012), and the methods for amplification of tufA are described in Fama et al. (2002). The ITS region (including the 5.8S gene) was amplified using primers and conditions from White et al. (1990) and Shoup and Lewis (2003). Sequence reads were assembled into contigs using either Sequencher ver. 4.5 (GeneCodes Inc., Ann Arbor, MI, USA) or Geneious ver. 5.4 (Biomatters Ltd., Auckland, New Zealand). GenBank accession numbers are provided in Table 1. Taxon selection was based on previous studies on Sphaeropleales as well as preliminary, more inclusive analyses, and was designed to include 1–2 representatives of genera morphologically similar to Bracteacoccus to demonstrate that the strains of concern represent distinct lineages. Other sphaeroplealean genera were selected for the data set to achieve even sampling within the order and especially a sampling as complete as possible within the clades containing Bracteacoccus, Follicularia, Planktosphaeria, and Pseudomuriella.

To confirm the monophyly of Sphaeropleales and to establish plausible rooting for the within-Sphaeropleales analyses, we conducted a Phycas analysis of three chloroplast genes (psaB, psbC, and rbcL, partitioned by gene and codon position) including representatives of other chlorophycean orders (Chaetophorales, Chaetopeltidales, Oedogoniales, and Volvocales, Table S1). The resulting tree (Fig. S1 in the Supporting Information) was consistent with Tippery et al. (2012), in that the clade comprising Chaetopeltidales, Chaetophorales, and Oedogoniales was sister to the remaining Chlorophyceae, and Volvocales was the sister taxon to Sphaeropleales.

Within-Sphaeropleales phylogenetic analyses

All DNA sequences were aligned manually and regions of uncertain homology in rDNA were excluded from all analyses. The concatenated 7-gene data set was subjected to a series of stepping-stone analyses (Fan et al. 2011) using Phycas v.1.2 (Lewis et al. 2011) to identify the best partitioning scheme from the examined six (Table 2) by comparing estimated marginal likelihoods. A GTR+I+G model was applied to each subset regardless of partitioning strategy. Phycas stepping-stone analyses involved 10,000 cycles of a single Markov chain for each of 21 beta values. An additional 20,000 cycles were added at the beginning (beta = 1) to ensure adequate parameterization of the reference distribution. The tree topology was constrained to the one shown in Figure 2 for all stepping-stone analyses. The second-most complex partitioning scheme scored best, and therefore the data set was divided into 13 subsets: rDNA (18S, 5.8S, and 28S), and each protein-coding gene divided by codon position (rbcL 1st positions, rbcL 2nd positions, rbcL 3rd positions, tufA 1st positions, etc.).

Table 2. Comparison of the partitioning schemes considered for analysis of the 7-locus data set tested with the steppingstone method, and their marginal likelihood scores. All subset partitions were analyzed under the GTR + I + G model of evolution
Partitioning scheme# of subsetsMarginal likelihood ScoreDifference from best scheme
By compartment (rDNA vs. plastid)2−70141.563294.63
By gene7−69898.103051.17
Plastid by codon, rDNA by gene6−66892.2945.36
By gene and codon15−66847.770.84
By gene and codon, rDNA combined13−66846.93Best

A maximum likelihood (ML) analysis was conducted on the partitioned (by gene and codon position) 7-gene data set using Garli v.2.0 (Zwickl 2006), with five independent searches for the best tree and 100 bootstrap (BS) pseudoreplicates to estimate branch support. In addition to a combined partitioned analysis, each gene was analyzed separately using Phycas to assess phylogenetic signal coming from individual data subsets. In the cases of protein-coding genes, the data sets were partitioned by codon position. Similarly, phylogenetic signal from nuclear genes versus plastid genes was compared by analyzing these subsets of data separately, with plastid genes partitioned by gene and codon position. All Phycas analyses were run for 100,000 cycles with polytomies allowed, and the first 200 cycles were discarded as burn-in. Phycas scripts specifying settings and priors used are provided in the supplementary materials Appendix S1 in the Supporting Information.

Because this is a study of taxa that have already proven difficult to place phylogenetically, we used Bayesian Concordance Analysis (BCA; Ané et al. 2007) to investigate the degree of phylogenetic concordance amongst the seven genes. Complete concordance means all genes share the same tree topology, while complete discordance means each gene evolved on a unique tree topology. Unlike other species tree approaches, BCA makes no assumptions about the underlying causes of discordance, using nonparametric Bayesian clustering to estimate the posterior distribution of gene-tree maps, which map each gene to a particular tree topology. BUCKy (Larget et al. 2010) was used to carry out BCA. BUCKy uses samples from the posterior distributions of single-gene analyses as input, but does not allow polytomies, so separate single-gene analyses (that did not consider polytomous trees) were performed in Phycas only for BCA.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Supporting Information


The newly characterized strains UTEX B2977, SAG 2265, BCP-ZNP1VF31, and UTEX B2979 resemble members of the genus Bracteacoccus morphologically (Fig. 1). Vegetative cells are spherical to somewhat irregular, and young cells (Fig. 1, a, k and m) have a single-lobed parietal chloroplast, which during maturation fragments into multiple chloroplasts lacking sheathed pyrenoids. TEM was not used, and therefore the presence of naked pyrenoids cannot be ruled out in these taxa. In UTEX B2977 and SAG 2265, plastids are small and numerous in mature cells (Fig. 1, g, n and o), which are clearly multinucleate (Fig. 1, l and p). Mature cells of UTEX B2979 have fewer, larger chloroplasts (Fig. 1, d–f) that sometimes appear layered (Fig. 1c). Multinuclearity is less obvious in this strain (Fig. 1b). All stages of strain BCP-ZNP1VF31 could not be examined in detail because the culture died during the progress of this study. Cell walls do not thicken appreciably with age in any of the examined isolates (Fig. 1).


Figure 1. Cells of Rotundella rotunda (a–f), Tumidella tumida (g–l), and Bracteamorpha trainorii (m–r) observed using light microscopy, representing ranges of morphologies in each taxon from young cells with a single lobed chloroplast per cell to mature polyplastidic cells with multiple nuclei (arrows in b, l, p) to flagellated cells (where available, h, i, r). (a, k, m) Young vegetative cells. (c) Autospore production, young cell with an unusual layered chloroplast morphology. (d, e, g, n, o) Maturing polyplastidic cells. (f, j, l, p, q) Aging cells accumulating carotenoid pigments. (h, r) Asexual zoospore, (i) quadriflagellate cell after syngamy. Scale bar in (m) represents 5 μm and applies to all images except flagellated cells.

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In all strains studied, older cells accumulate secondary carotenoids (Fig. 1, f, j and q). Older cultures are orange in color (UTEX B2977, SAG 2265) or orange-brown (UTEX B2979). All three strains reproduce asexually by way of autospores (e.g., Fig. 1c). Production of biflagellate naked zoospores was observed in UTEX B2977 and SAG 2265 (Fig. 1, h and r), and previously reported in relatives of UTEX B2979 (Flechtner et al. 2013). In UTEX B2977, zoospore flagellar length appeared slightly unequal and the stigma, often difficult to observe, was anterior. Quadriflagellate cells at various stages of fusion/separation occurred frequently, but the process of cell fusion was not observed directly. In SAG 2265, zoospores were of highly variable shapes ranging from slender and elongate, sometimes with a posterior protrusion, to pyriform or ovoid, sometimes flattened. Flagellated cells were observed to either settle after a few minutes of swimming, or function as gametes. Here, pairs of cells initially touched at their anterior ends, and subsequently fused in a matter of minutes, resulting in large quadriflagellate cells of various shapes (Fig. 1i). A stigma was observed only in a few biflagellate cells and was either median or slightly posterior. Relative flagellar length was difficult to assess, but in the few cases where flagella aligned next to each other, they appeared equally long.

Molecular analyses

The pan-Chlorophyceae analysis showed the genus Mychonastes as sister to the remaining Sphaeropleales (Fig. S1), and we used this information to root the trees resulting from all subsequent analyses, although the actual relationships among sphaeroplealean families remain unresolved. The full within-Sphaeropleales data set had 8,916 nucleotides, 5,049 from the chloroplast genes, and 3,867 from the nuclear ribosomal genes. After pruning 129 rDNA sites of uncertain homology, 8,787 sites remained. This final data set was 92.9% complete, with the majority of missing data located at the 5′ and 3′ ends of individual genes. In addition, we were able to collect only partial data for several taxa. At the 5′ end of 28S, 581 bp were missing in Pseudomuriella engadinensis (Kol & F. Chodat) Fučíková, Rada & L. A. Lewis (UTEX 58), Follicularia botryoides (Herndon) Komárek (UTEX LB951), and Rotundella sp. (BCP-ZNP1VF31). In Follicularia texensis (H. W. Bischoff & H. C. Bold) H. Ettl & Komárek (UTEX 1241) only 58 out of the total 154 bp of 5.8S were collected and no 5.8S data were obtained for Chlorotetraedron incus (Teiling) Komárek & Kováčik (SAG 43.81) and Characiopodium hindakii (K. W. Lee & H. C. Bold) G. L. Floyd & Shin Watan. (UTEX 2098). Ourococcus multisporus H. W. Bischoff & H. C. Bold (UTEX 1240) was missing 598 bp at the 5′ end of tufA and Kirchneriella aperta Teiling (SAG 2004) was missing 363 bp at the 3′ end. No tufA data were collected for Botryosphaerella sudetica (Lemmermann) P. C. Silva (UTEX 2629), Characiopodium hindakii (UTEX 2098), Mychonastes jurisii (Hindák) Krienitz, C. Bock, Dadheech & Pröschold (SAG 37.98), and Parapediastrum biradiatum (Meyen) E. Hegewald (UTEX 37). No psbC data were obtained for Rotundella sp. (BCP-ZNP1VF31).

The 18S data set comprised 1,687 characters after exclusion of 89 sites of dubious homology. The 28S data set comprised 1,897 characters after exclusion of 40 sites of dubious homology, and the 5.8S data set comprised 154 characters with no excluded sites. The rbcL data set comprised 1,290 sites, the psbC data set 1,089 sites, the psaB data set 1,785 sites, and the tufA data set 885 sites. Alignments are available from (study 13960).

Bayesian phylogenetic analyses of individual genes where polytomous trees were allowed (Fig. S2 in the Supporting Information) revealed conflict in the backbone of the tree (poorly supported for the most part). Figure 2 shows the BCA concordance tree based on single-gene analyses, but also presents the results of our combined partitioned analyses by indicating both Bayesian posterior probabilities and BS values in addition to the concordance factors for all nodes. In general, shallower nodes, corresponding to existing and proposed families in our study, were well supported by both ML and Bayesian analyses, and also often received high concordance factor values. The best ML tree and the Bayesian consensus tree had identical topologies and were similar to the concordance tree, except for the backbone. All previously established families were recovered as monophyletic (Bracteacoccaceae, Hydrodictyaceae, Neochloridaceae Radiococcaceae, Scenedesmaceae, Selenastraceae, and Sphaeropleaceae) and were well to moderately supported (Fig. 2). The separate rDNA and plastid analyses yielded trees with most disagreement in the backbone, but otherwise largely congruent (Fig. S3 in the Supporting Information). Notably, Neochloridaceae received good support from the rDNA data, but was not monophyletic in the plastid gene analysis (Fig. S3).


Figure 2. Concordance tree based on Bayesian analyses of seven single-locus data sets (18S, 28S, 5.8S, psaB, psbC, rbcL, and tufA) and covering the order Sphaeropleales. Rooting is based on the broader analysis shown in Fig. S1. Strains representing the newly described taxa are highlighted in larger font. Numbers above branches indicate statistical support for nodes: Bayesian posterior probabilities (BPP) and maximum likelihood bootstrap values (BS), respectively. Numbers below branches indicate concordance factors. Values of 1.00 and 100 are represented by asterisks (*) and values lower than 0.50 and 50% are represented by dashes (-). Scale bar represents expected number of substitutions per site. Newly proposed family names are indicated in black font on the right-hand side, and previously recognized families are indicated in gray font. Reproduction via flagellated cells is indicated for those families where it has been reported previously. Icons depict the known distribution of flagellated cells across families in Sphaeropleales. Filled icons indicate families for which both zoospores and gametes are known; unfilled icons represent presence of asexual zoospores only. Schizochlamydaceae is presented as asexual because taxonomic placement of the putative sexual strain of Schizochlamys cannot be confirmed.

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No single gene yielded a fully resolved topology, and large polytomies were found in the 18S, rbcL, and tufA consensus trees. Neochloridaceae was recovered as monophyletic only in the 28S and tufA phylogenies. The remainder of the single-locus analyses positioned Chlorotetraedron either as sister to Hydrodictyaceae or as sister to Hydrodictyaceae + Neochloridaceae. The remaining established sphaeroplealean families were recovered as monophyletic in all single-gene phylogenies, with the exceptions of Radiococcaceae (para- or polyphyletic in 28S, psbC, rbcL, and tufA) and Scenedesmaceae (polyphyletic in psbC). The coccoid clade comprising Bracteacoccaceae, Bracteamorphaceae, Schizochlamydaceae, Radiococcaceae, and Tumidellaceae was only recovered as monophyletic in the 28S phylogeny (Fig. S2).

The newly isolated BCP desert strains UTEX B2977, UTEX B2979, ZNP1VF31, and the SAG strain 2265 formed three deeply divergent lineages distinct from any genus or family recognized to date (Fig. 2, Figs. S1 and S2). The isolates UTEX B2979 and ZNP1VF31 appeared closely related and formed a well-supported clade that was sister to Scenedesmaceae in the multilocus analyses. Likewise, in the rDNA and the plastid DNA consensus trees, this clade was strongly supported as separate from other Bracteacoccus-like lineages. In the full seven-gene analyses, SAG 2265 and UTEX B2977 were resolved as members of the clade containing the families Bracteacoccaceae, Schizochlamydaceae, and Radiococcaceae (Fig. 2). The analysis of chloroplast genes resolved these two strains as a poorly supported clade that was weakly supported as sister to Bracteacoccus. In the rDNA-only analysis, these two strains formed a basal grade in the group of Bracteacoccaceae, Radiococcaceae, and Schizochlamydaceae.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Supporting Information

Coccoid lineages in Sphaeropleales

On the basis of our results (Fig. 2), we divide the order Sphaeropleales into 17 families, three of which accommodate newly discovered lineages. Eleven of the seventeen families solely comprise simple spherical, ellipsoidal, or ovoid unicells or loose colonies of such cells. Among the remaining six, Neochloridaceae and Scenedesmaceae also contain a number of coccoid taxa. Schroederiaceae, Selenastraceae, and Sphaeropleaceae all contain vegetatively nonmotile unicells of various shapes, although many Selenastraceae form loose groups/colonies. Obligate colonies with defined numbers of cells (coenobia) only occur in the Hydrodictyaceae and Scenedesmaceae. This prevalence of coccoid forms is remarkable—the order Sphaeropleales is mostly known for the morphologically complex Hydrodictyaceae and Scenedesmaceae, which have received a great deal of taxonomic attention in the past and contain hundreds of species and varieties. Here, we demonstrate that complex morphologies in Sphaeropleales are phylogenetically restricted, while most of the genetic diversity is found in deeply diverging coccoid lineages.

Among the multitude of green coccoid soil algae, Bracteacoccus appears relatively easy to distinguish, possessing multiple nuclei and discoid parietal chloroplasts in mature cells, which are more or less spherical (Ettl and Gärtner 1995). However, recent findings demonstrated the existence of other sphaeroplealean lineages possessing the same overall morphology, namely the genera Chromochloris and Pseudomuriella (Fučíková et al. 2011a, Fučíková and Lewis 2012). Depending on taxon sampling and marker used, these genera have been resolved at different positions within Sphaeropleales. Some analyses suggested the existence of a larger clade uniting spherical coccoids with multiple plastids and nuclei (Fučíková and Lewis 2012 fig. 17). However, the present study illustrates the presence of other, deeply diverging sphaeroplealean coccoid lineages, suggesting that the diversity of these inconspicuous yet common soil inhabitants is still severely underestimated. Moreover, this study shows that Bracteacoccus-like lineages are not aggregated in a single clade (Fig. 2), but rather dispersed throughout Sphaeropleales. Specifically, the clades containing the genera Pseudomuriella, Chromochloris, and the newly proposed Rotundella do not form a monophyletic group with the coccoid families Bracteacoccaceae, Radiococcaceae, and the newly proposed Bracteamorphaceae and Tumidellaceae. Additionally, Bracteacoccus-like algae appear to be especially successful in soil habitats, although it is not immediately obvious what aspect of this particular morphotype (other than the accumulation of protective carotenoid pigments and the physical advantages of spherical cells) equips them for a terrestrial life style.

Sexual reproduction in Sphaeropleales

To date, sexual reproduction has been documented only in a handful of sphaeroplealean lineages (Fig. 2). Wilcox and Floyd (1988) described the ultrastructure of Pediastrum gametes, which appeared similar to asexual zoospores except for the possession of the apical mating structure, an eyespot, and the lack of cytoskeletal features involved in colony formation. Gametogenesis and syngamy are very rare in the Scenedesmaceae, but Scenedesmus gametes were induced and the conditions optimal for their production were reported by Trainor and Burg (1965) and later by Cain and Trainor (1976). Přibyl and Cepák (2007) reported fusion of unusual quadriflagellate gametes in Botryosphaerella sudetica (Neochloridaceae). Anisogamy or oogamy with heteromorphic male and female gametes occurs in the Sphaeropleaceae (e.g., Cáceres et al. 1997). Sexual reproduction was also described for Schizochlamys gelatinosa A. Braun (Thompson 1956), but it is impossible to determine whether or not the strain used in 1956 was closely related to SAG 66.94. Thus, our report of sexual reproduction in the newly characterized lineages Bracteamorphaceae and Tumidellaceae is a very important finding. Syngamy was not observed in UTEX B2977, but the entire process was followed in SAG 2265. Since quadriflagellate cells were observed in both strains, it is quite possible that both are capable of sexual reproduction. As the observed gametes were of similar morphologies, the type of mating can be described as isogamy. It is notable that sexual reproduction was never reported in Bracteacoccus, Follicularia, Planktosphaeria, or Radiococcus, although zoospore production is well known from most of these genera. In light of our findings, it is possible that sex is more common in Sphaeropleales than previously thought, but only occurs under specific environmental conditions.

Family-level taxonomy in Sphaeropleales

The family Neochloridaceae contains aquatic coccoid algae that are mostly multinucleate, spherical or of more intricate polyhedral shapes, and have pyrenoids surrounded by continuous starch sheaths without thylakoid invaginations (e.g., Watanabe et al. 1988). Asexual reproduction happens via aplanospores or naked or fuzzy biflagellate zoospores that have been studied using TEM in Chlorotetraedron (Watanabe et al. 1988), Characiopodium (Floyd et al. 1993), and Neochloris (Watanabe and Floyd 1989). The ultrastructure of cell division was described for Neochloris (Kouwets 1995). In this study, Neochloridaceae were represented by the type genus Neochloris, the genus Characiopodium, and the genus Chlorotetraedron to capture the most phylogenetic diversity possible within the family (Hegewald et al. 2001). Additionally, “Botryococcussudeticus has been shown to be neochloridacean and thus separate from the authentic trebouxiophycean Botryococcus (Senousy et al. 2004, confirmed in the present study). Even prior to the phylogenetic study of Senousy et al. (2004), this species was recombined into Botryosphaerella, although this transfer has been acknowledged rarely in practical use (Silva 1970). Botryosphaerella sudetica forms clusters, but is not as clearly colonial as true Botryococcus species. In the present study, Neochloridaceae was monophyletic in the 28S and tufA analyses. The placement of the deepest-diverging taxon, Chlorotetraedron in Neochloridaceae was weakly contradicted in some single-locus analyses (Fig. 2 and Fig. S2). A study by Hegewald et al. (2001) determined that Polyedriopsis also belongs to this family.

Another coccoid genus, Mychonastes, recently underwent a taxonomic revision. Phylogenetic analyses presented in Krienitz et al. (2011) indicated that this genus represents a divergent lineage distinct from any family recognized to date. Tsarenko (2005) placed Mychonastes in Scotiellocystoidaceae, but that classification was rather confusing, as the proposed family contains members of Scenedesmaceae (Scotiellopsis, Graesiella) as well as other genera of unknown affiliation (e.g., Halochlorella, Muriellopsis), and is classified within the order Chlorococcales, the polyphyly of which had been established prior to Tsarenko (2005; e.g., Lewis et al. 1992, Wilcox et al. 1992). Our analyses suggested that Mychonastes may be the deepest-diverging lineage of Sphaeropleales (Fig. S1). Using a phylogenetic approach to taxonomy, we propose a new monotypic family Mychonastaceae to accommodate this genus currently comprising 20 valid species, ten of which have been validated with molecular data (Krienitz et al. 2011). The Mychonastaceae are aquatic uninucleate coccoid algae lacking pyrenoids, with no known flagellated stages. Mychonastes homosphaera (Skuja) Kalina and Punčochářová has been examined using EM and shown to have a network of fine ribs on the cell surface (Kalina and Punčochářová 1987).

Similarly to the case above, we propose a monotypic family Dictyococcaceae to accommodate the genus Dictyococcus with its only known species D. varians Gerneck. This alga is aquatic, multinucleate, polyplastidic with no pyrenoids, and reproduces asexually via naked biflagellate zoospores with equally long flagella (Starr 1955). The historical taxonomic confusion between Dictyococcus and Bracteacoccus was resolved in Fučíková et al. (2011a).

The family Selenastraceae, represented here by Ankistrodesmus falcatus (Corda) Ralfs, Kirchneriella aperta, and Ourococcus multisporus, was recovered as monophyletic with good statistical support in all of our analyses, and requires no taxonomic update. This family contains aquatic, uninucleate, fusiform or sickle-shaped algae that are either solitary or colony-forming. Chloroplasts often contain pyrenoids that are either starch-covered or naked, often with thylakoid invaginations (Krienitz et al. 2001). No flagellated stages have been reported in this family. The polyphyly of Ankistrodesmus and Monoraphidium was demonstrated in both Krienitz et al. (2001) and Fawley et al. (2006), but formal revisions have yet to be made. Other representatives of the family are Selenastrum, Podohedriella, Quadrigula, and Raphidocelis.

The monophyly of the aquatic and coenobial Hydrodictyaceae was consistently recovered in our analyses. This family was treated in detail by Buchheim et al. (2005), who erected several new genera. More recently, McManus and Lewis (2011) examined this family, emphasizing the genus Pediastrum, with the use of both molecular analyses and inspection of surface structures of many taxa using SEM. The status of the type genus has not yet been resolved, and Hydrodictyon currently remains nested within Pediastrum. Hydrodictyaceae have a single plastid and pyrenoid per cell (with the exception of Hydrodictyon, which has many pyrenoids and a reticulate plastid), multiple nuclei, and they reproduce asexually via autospores or zoospores, or sexually via isogamous biflagellate gametes. The development of flagellated cells of Pediastrum was described by Hawkins and Leedale (1971), and their ultrastructure by Wilcox and Floyd (1988).

Scenedesmaceae contains numerous coenobial species of Desmodesmus, Neodesmus, and especially Scenedesmus, although some representatives of the last named genus are only known in solitary coccoid form (e.g., S. rotundus L. A. Lewis & Flechtner used in the present study). Other genera in this family are either coenobial (e.g., Coelastrum, Hariotina) or solitary (e.g., Scotiellopsis) and often have elaborate cell wall ornamentation (Kalina and Punčochářová 1987). Scenedesmacean algae generally have one nucleus and a single plastid with a pyrenoid in each cell, and reproduce asexually via autospores or zoospores, or rarely sexually via isogamy (Cain and Trainor 1976). The most recent genus-level taxonomic treatment of this family was presented in Hegewald et al. (2010).

The lineage resolved as sister to Scenedesmaceae, Neochloridaceae, and Hydrodictyaceae comprises two desert soil crust isolates, for which we propose a new genus name, Rotundella. Only UTEX B2979 remains alive to date, and for this strain, we propose a new species name R. rotunda. We also erect a new monotypic family, Rotundellaceae, to accommodate this lineage that currently only comprises coccoid soil inhabitants with rarely occurring asexual flagellated stages.

Pseudomuriella also does not appear associated with any recognized family in Sphaeropleales. The genus currently comprises four cryptic species, and the genetic diversity within this genus was described in Fučíková et al. (2011b). We erect the family Pseudomuriellaceae to harbor this lineage. Pseudomuriella is a multinucleate, polyplastidic alga that lacks pyrenoids and may easily be confused with Bracteacoccus. While zoospores have been reported in Pseudomuriella (Kalina and Punčochářová 1987), they have not been examined in detail using EM.

The fusiform aquatic genus Schroederia, represented in this study by the type species S. setigera (Schröder) Lemmermann, is currently classified as a member of Characiaceae. However, this family is now known to be polyphyletic, containing members of Sphaeropleaceae (Ankyra) as well as Neochloridaceae (Characiopodium), and volvocalean algae such as Characium and Chlamydopodium (reviewed in Lewis and McCourt 2004). Our results suggest that Schroederia should be placed in a separate family, for which we propose the name Schroederiaceae. This family likely also includes the genus Pseudoschroederia (relationship with Schroederia shown in Shoup and Lewis 2003). The ultrastructure and reproduction of Schroederia and Pseudoschroederia were examined by Hegewald and Schnepf (1986), who described the algae as uninucleate, monoplastidic, and reproducing asexually via autospores or biflagellate zoospores. Pyrenoids in this family are enclosed in starch sheaths and are not invaginated by thylakoids.

A morphology-based revision of the Radiococcaceae was published by Kostikov et al. (2002), and included a number of genera. Of these, our study only included representatives of Radiococcus and Planktosphaeria. Figure 2 shows that the sister group to Radiococcus is the genus Follicularia, which we herein include in Radiococcaceae. Kostikov et al. (2002) also included Schizochlamydella in this family, but it was since shown to be closely related to the trebouxiophyte Oocystis (Wolf et al. 2003). Planktosphaeria grouped strongly with Schizochlamys, but because a considerable phylogenetic distance separates Radiococcaceae and these two genera and because their sister relationship was only weakly supported, we propose a new family name, Schizochlamydaceae, for the clade containing Planktosphaeria and Schizochlamys. Both Radiococcaceae and Schizochlamydaceae are coccoid or loosely colonial, possibly monadoid in the case of Schizochlamys, uni- or multinucleate, mono- or polyplastidic, contain pyrenoids, and are able to produce biflagellate zoospores.

Bracteacoccaceae was erected by Tsarenko (2005), who included Planktosphaeria in this family along with Bracteacoccus. As discussed above, Planktosphaeria falls within another clade, the herein proposed Schizochlamydaceae. The database lists Chromochloris as a member of the Bracteacoccaceae, but this inclusion was not supported by our analyses. We propose that Bracteacoccus, at present, be the only genus in Bracteacoccaceae. Bracteacoccaceae are terrestrial coccoids that reproduce via aplanospores or biflagellate zoospores with unequal flagella. Their ultrastructure was studied by Kouwets (1993, 1996 – cell cycle) and Watanabe and Floyd (1992 – zoospores).

The coccoid strain SAG 2265 was isolated from the Namib desert and while morphologically very similar to other Bracteacoccus-like algae, phylogenetically appeared very distinct in all our analyses. We therefore propose a new genus name for it, Tumidella. The desert strain UTEX B2977, isolated from Carlsbad Caverns, NM represents a new, distinct Bracteacoccus-like lineage, for which we suggest the genus name Bracteamorpha. The two genera are genetically very divergent from one another, and from all other genera included in this study. They are morphologically similar to one another and their relatives, but stand out, in that they appear capable of sexual reproduction, unlike any of their close relatives. Because their relationship as sister taxa was not recovered in most analyses (Fig. 2, Fig. S2), we propose two new family names to accommodate these divergent lineages: Bracteamorphaceae and Tumidellaceae. Our analyses suggest that Bracteacoccaceae, Bracteamorphaceae, Radiococcaceae, Schizochlamydaceae, and Tumidellaceae form a clade of mostly coccoid coenocytic algae with multiple chloroplasts per cell, mostly capable of zoospore production. However, as discussed above, other Bracteacoccus-like algae are found outside of this clade: Chromochloris, Pseudomuriella, and Rotundella.

The genus Chromochloris was resurrected by Fučíková and Lewis (2012) and currently contains one species, C. zofingiensis (Dönz) Fučíková & L. A. Lewis. According to our multi-locus analyses, Chromochloris represents a lineage distinct from any recognized family, and we therefore establish Chromochloridaceae to harbor this genus. Chromochloris is morphologically similar to Bracteacoccus, as it is polyplastidic and multinucleate, lacks pyrenoids, and produces biflagellate zoospores. Its vegetative ultrastructure was described in Kalina and Punčochářová (1987).

Likewise, the genus Dictyochloris represents another early diverging sphaeroplealean lineage that clearly falls outside of Radiococcaceae, wherein it currently is classified. We therefore propose the Dictyochloridaceae to accommodate this taxon. The spherical coccoid Dictyochloris is characterized by its reticulate chloroplast and biflagellate zoospores ultrastructurally similar to Bracteacoccus due to their parallel flagellar basal bodies (Watanabe and Floyd 1992).

The type family Sphaeropleaceae differs from many other sphaeroplealeans in ITS2 secondary structure (Keller et al. 2008), although most lineages discussed in the present study have not been tested in that way. It is possible that Sphaeropleaceae are sister to the remaining (“crown”) Sphaeropleales as suggested by previous studies (e.g., Wolf et al. 2002, Tippery et al. 2012). The order Sphaeropleales is characterized by a putatively strong ultrastructural synapomorphy, the directly opposed (DO) basal flagellar bodies in motile cells, and even though swimming cells of the Sphaeropleaceae differ ultrastructurally from the remainder of the order, they retain the basic DO organization. The Sphaeropleaceae contain the filamentous Sphaeroplea, and the solitary Ankyra and Atractomorpha. These algae are coenocytic, contain pyrenoids with traversing cytoplasm, and reproduce via zoospores or sexually via anisogamy or oogamy. Motile cells have been described in detail by Hoffman (1984) and Cáceres et al. (1997).

In addition, Tsarenko (2005) lists four families as members of the Sphaeropleales that were not included in our study: Characiaceae, Chlorosarcinaceae, Gloeotilaceae, and Microsporaceae. Characiaceae, as discussed above, is a problematic taxon because the higher taxonomic affiliation of the lectotype species (Characium sieboldii A. Braun) remains unclear. Nevertheless, the classification proposed in this study is not affected by this uncertainty. The family Chlorosarcinaceae was not considered in this study because it had been demonstrated by Deason and Floyd (1987) that Chlorosarcina has a counter-clockwise arrangement of flagellar basal bodies, which rules it out as a member of Chlorophyceae. Similarly, even though the type of the genus has not been verified phylogenetically, Gloeotila was shown to group with trebouxiophytes in Verghese (2007). Likewise, no type material of Microspora is available for phylogenetic examination, and therefore the family was not considered in the present study. Interestingly, unpublished data by Buchheim and Buchheim ( weakly suggest that one Microspora-like strain (UTEX LB472) may be a relative of Sphaeropleales. However, Durako (2007) showed the same strain as a relative of Chaetophorales. The phylogenetic position of Microsporaceae will require further investigation, and likely additional field collections to designate a type specimen/culture for the generitype of Microspora, M. abbreviata (Rabenhorst) Lagerheim.

Taxonomic revisions

Rotundella rotunda gen. et sp. nov. Fučíková, P. O. Lewis & L. A. Lewis (Fig. 1, a–f)

Cells spherical to ovoid or irregular (5–) 8–20 μm in diameter. In young cells, chloroplast single, cup-shaped or lobed, and parietal; at maturity, chloroplasts multiple but large and few in numbers, parietal. Mature cells multinucleate. Sheathed pyrenoid absent but starch grains present. Cell wall thin and smooth. Oil droplets and pigments accumulating in aging cells. Old cultures orange-brown. Asexual reproduction via autospores or naked biflagellate zoospores; sexual reproduction not observed. Genus differentiated from other taxa in Sphaeropleales by 18S rRNA and rbcL gene sequences.

Holotype: Specimen CONN00177433.

Isotype: Culture UTEX B2979, University of Texas, Austin, TX, USA

Type locality: Joshua Tree National Park, CA, USA

Tumidella tumida gen. et sp. nov. Fučíková, P. O. Lewis & L. A. Lewis (Fig. 1, g–l)

Cells spherical to ovoid or irregular, 5–33 μm in diameter. In young cells, chloroplast single, lobed and parietal. At maturity, chloroplasts small and numerous, both parietal and internal. Sheathed pyrenoid absent. Mature cells noticeably multinucleate, nuclei scattered throughout the cell's volume. Cell wall thin and smooth. Golden or orange pigment accumulating in aging cells. Asexual reproduction via autospores (mostly 8 or 16 per mother cell) or biflagellate naked zoospores. Zoospores of variable shapes and sizes, ranging from very elongate and slender (2 × 15–19 μm) to shorter, pear shaped (3.3–4 × 6–8 μm), or sometimes dorsoventrally flattened and wide (up to 6.5 μm). Prominent anterior vacuole; stigma mostly not visible, median or slightly posterior when observable. Two flagella of equal length. Zoospores either settle and become vegetative cells after losing flagella, or function as gametes and fuse to form quadriflagellate zygotes. Genus differentiated from other taxa in Sphaeropleales by 18S rRNA and rbcL gene sequences.

Holotype: Specimen ONN00177865

Isotype: culture SAG 2265

Type locality: Namib Desert, Namibia.

Bracteamorpha trainorii gen. et sp. nov. Fučíková, P. O. Lewis & L. A. Lewis (Fig. 1, m–r)

This species is named after the late Dr. Francis R. Trainor, phycologist and Professor Emeritus, University of Connecticut.

Cells spherical to irregularly ovoid, up to 14 μm wide and 24 μm long. In young cells chloroplast single, parietal and lobed. At maturity, chloroplasts numerous and small, both parietal and internal. Sheathed pyrenoid absent. Mature cells multinucleate. Cell wall thin and smooth, not thickening appreciably with age. Orange pigment accumulating in older cells. Asexual reproduction via autospores (4–16 per mother cell, up to 5 μm in diameter) or biflagellate naked zoospores. Zoospores elongated, 2.0–4.0 μm wide and 5–16 μm long. Light orange pigment masking zoospore nucleus; stigma small and anterior. One or two chloroplasts per zoospore present. Two flagella of slightly uneven length. Frequent quadriflagellate cells indicating sexual reproduction. Genus differentiated from other taxa in Sphaeropleales by 18S rRNA and rbcL gene sequences.

Holotype: CONN00177434

Isotype: Culture UTEX B2977, University of Texas, Austin, TX, USA

Type locality: Carlsbad Caverns National Park, Eddy Co., New Mexico, USA

Bracteamorphaceae fam. nova

Soil-dwelling spherical coccoids with multiple chloroplasts lacking pyrenoids; multinucleate. Asexual reproduction via autospores, aplanospores, or biflagellate naked zoospores. Sexual reproduction via isomorphic biflagellate zoospore-like gametes.

Type genus: Bracteamorpha

Rotundellaceae fam. nova

Soil-dwelling spherical coccoids with multiple chloroplasts lacking pyrenoids; multinucleate. Asexual reproduction via autospores, aplanospores or rarely biflagellate zoospores. Sexual reproduction not known.

Type genus: Rotundella

Tumidellaceae fam. nova

Soil-dwelling spherical coccoids with multiple chloroplasts lacking pyrenoids; multinucleate. Asexual reproduction via autospores, aplanospores, or biflagellate naked zoospores. Sexual reproduction via isomorphic biflagellate zoospore-like gametes.

Type genus: Tumidella

Pseudomuriellaceae fam. nova

Soil-dwelling spherical coccoids with multiple chloroplasts lacking pyrenoids; multinucleate. Asexual reproduction via autospores, aplanospores, or biflagellate naked zoospores. Sexual reproduction not known.

Type genus: Pseudomuriella

Dictyococcaceae fam. nova

Spherical aquatic coccoids with multiple chloroplasts with inflected edges, lacking pyrenoids; multinucleate. Asexual reproduction via autospores or biflagellate naked zoospores. Sexual reproduction not known.

Type genus: Dictyococcus

Mychonastaceae fam. nova

Spherical, ovoid, or ellipsoidal aquatic coccoids either solitary or colonial. One to four chloroplasts per cell, lacking pyrenoid; uninucleate. Asexual reproduction via autospores. Sexual reproduction not known.

Type genus: Mychonastes

Schroederiaceae fam. nova

Solitary aquatic algae, elongate with apical protrusions. Chloroplasts single or multiple before reproduction, with one or more pyrenoids; at maturity multinucleate. Asexual reproduction via autospores or biflagellate zoospores. Sexual reproduction not known.

Type genus: Schroederia

Schizochlamydaceae fam. nova

Aquatic algae, solitary or in small aggregations, coccoid or flagellate with multiple chloroplasts; pyrenoids present; at maturity multinucleate. Asexual reproduction via autospores or biflagellate zoospores. Sexual reproduction not known.

Type genus: Schizochlamys

Chromochloridaceae fam. nova

Soil-dwelling spherical coccoids with multiple chloroplasts lacking pyrenoids; multinucleate. Asexual reproduction via autospores, aplanospores or biflagellate zoospores. Sexual reproduction not known.

Type genus: Chromochloris

Dictyochloridaceae fam. nova

Terrestrial spherical coccoids with a net-like chloroplast lacking pyrenoids; multinucleate. Asexual reproduction via autospores or biflagellate zoospores. Sexual reproduction not known.

Type genus: Dictyochloris

This work was supported by the NSF grant DEB-1036448 (Assembling the Green Algal Tree of Life: GrAToL) awarded to L. A. Lewis and P. O. Lewis. We thank Dr. V. Flechtner from John Carroll University for the initial morphological observations on the strain UTEX B2977, and Dr. N. Pietrasiak for isolating and initial characterization of the strain UTEX B2979.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Supporting Information
jpy12118-sup-0001-FigS1.pdfapplication/PDF182KFig. S1. Bayesian majority rule consensus tree based on the combined partitioned data set of rbcL, psaB, and psbC (4164 characters total) including taxa outside of Sphaeropleales. Scale bar represents expected number of substitutions per site. BPP values of 1.0 are represented by asterisks (*) and values lower than 0.50 are represented by dashes (-).
jpy12118-sup-0002-FigS2.pdfapplication/PDF335KFig. S2. Single-gene Bayesian majority rule consensus trees based on analyses of 18S, 28S, tufA, rbcL, psbC, and psaB, respectively. Scale bar represents expected number of substitutions per site. BPP values of 1.0 are represented by asterisks (*). Values lower than 0.50 are represented by dashes (-).
jpy12118-sup-0003-FigS3.pdfapplication/PDF292KFig. S3. Bayesian majority rule consensus trees based on combined rDNA data set (18S, 28S, 5.8S) and combined plastid data set (rbcL, psaB, psbC, and tufA) partitioned by gene and codon. Scale bar represents expected number of substitutions per site. Support values of 1.00 BPP and 100% BS are represented by asterisks (*). Values lower than 0.50 or 50% are represented by dashes (-). Branches shared between the two trees are highlighted with thicker lines.
jpy12118-sup-0004-TableS1.docxWord document17KTable S1. List of algal strains used as outgroup taxa for analysis presented in Fig. S1 with corresponding GenBank accession numbers for genes of interest. Sequences obtained for this study are highlighted in boldface font.
jpy12118-sup-0005-AppendixS1.zipZip archive197KAppendix S1. Phycas scripts specifying settings and priors used in this study.

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