Intersection of Chlorella, Muriella and Bracteacoccus: Resurrecting the Genus Chromochloris Ko L Et Ch O D a T (Chlorophyceae, Chlorophyta)
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Fottea 12(1): 83–93, 2012 83 Intersection of Chlorella, Muriella and Bracteacoccus: Resurrecting the genus Chromochloris KOL et CHODAT (Chlorophyceae, Chlorophyta) Karolina Fu č í k o v á * & Louise A. LEWIS University of Connecticut Department of Ecology and Evolutionary Biology, University of Connecticut, 75 North Eagleville Road, Storrs, 06269–3043 USA; *corresponding author e–mail: [email protected] Abstract: Coccoid green algae generally are difficult to classify because of their depauperate morphology, and for this reason many of them have a complicated taxonomic history. In the present study we use analyses of molecular sequence data to demonstrate the close relationship of two green coccoid taxa that have undergone multiple taxonomic transfers in the past, and argue for the recognition of a distinct lineage. We resurrect the genus Chromochloris to accommodate C. zofingiensis (= Muriella zofingiensis) and we provide data regarding the lineage’s phylogenetic relationships to other chlorophyte genera, especially Bracteacoccus, Chlorella, Muriella, Mychonastes, and Pseudomuriella. In addition, we synonymize B. cinnabarinus and Bracteacoccus minutus with C. zofingiensis, and clarify the identity of three UTEX strains heretofore listed as Bracteacoccus. Key words: 18S, Bracteacoccus cinnabarinus, cryptic taxa, ITS, rbcL, tufA Introduction transferred to the genus Dictyococcus Ge r n e c k by vi s c h e r (1936), and later into Bracteacoccus It is not uncommon for a single taxon te r e G by st a r r (1955). However, et t l & Gä r t n e r to undergo several transfers among two or more (1995) argued against accepting Bracteacoccus genera, for its name to be synonymized with cinnabarinus (ko l et ch o d a t ) st a r r because this another name and then subsequently be reinstated. species was described as uninucleate, whereas Coccoid green algae are notorious for their the genus Bracteacoccus is characterized by extremely confused taxonomic histories (LEWIS & multinucleate cells. To date, this alga is regarded Mcco u r t 2004), and more revisions are needed as a member of Bracteacoccus (Gu i r y & Gu i r y in order to accommodate new findings about their 2011) and the original culture of this species is diversity and systematic relationships. Viewed available from public culture collections (UTEX under a light microscope, the simple phenotypes 56, SAG 221–2). The systematic affinity of this of coccoid algae offer few traits for taxonomic use. taxon was the main focus of the present study. Two Even with morphological characters available, additional coccoid taxa of uncertain phylogenetic the interpretation of these characters is subjective position, B. minutus sc h w a r z and Muriella and different authors may consider different traits zofingiensis (dö n z ) hi n d á k , were examined in taxonomically important. DNA sequences offer order to determine their phylogenetic positions hundreds of characters and thus provide abundant within the Chlorophyceae. and independent data to evaluate evolutionary Bracteacoccus minutus, a species of histories of organisms, for which morphological a small size and containing numerous tiny characters are scarce, invariable or plastic, or chloroplasts, was described from soil from in which morphological homoplasy may be Dalmatia, Yugoslavia (now Croatia) (sc h w a r z common. 1979), without designation of type material. The genus Chromochloris as described by While this makes the name invalid, Schwarz’s ko l & ch o d a t (1934) comprises a single species original culture still exists and is available from Chromochloris cinnabarina ko l et ch o d a t , a soil the University of Innsbruck Culture Collection alga producing striking amounts of red carotenoid (ASIB S223). Therefore the taxonomic identity pigments in aging cultures. This species was of B. minutus can be verified and a type may be 84 Fu č í k o v á & LEWIS : The green coccoid genus Chromochloris designated if evidence is found for this strain to taxonomic histories and conflicting arguments represent a new, distinct species. about their affiliations, which in some cases are Muriella zofingiensis (strain SAG 211–14 further complicated by the absence of live type or its duplicates ATCC 30412 and UTEX 32) has material and/or improper typification. Our study been the target of several physiological studies is among the first contributions to green coccoid particularly due to its potential for secondary systematics that attempt to settle such taxonomic carotenoid production (e.g., ip & ch e n 2005; confusion through the use of molecular– hu a n G et al. 2006; LI et al. 2006). The taxonomic phylogenetic analyses (e.g., ne u s t u p a et al. 2009; and phylogenetic affiliation of this species also Bo c k et al. 2010; Šk a l o u d & pe k s a 2010; kr i e n i t z has received considerable attention. It was et al. 2011). originally described as Chlorella zofingiensis (dö n z 1934); then transferred into the genus Muriella by hi n d á k (1982) based on detailed Materials and Methods light microscopic morphological and life–cycle observations. In 1987, ka l i n a & punčochářová All examined cultures were grown on Bold’s Basal Medium (BBM, Bo l d 1949; Bi s c h o ff & Bo l d 1963) argued for the transfer of M. zofingiensis (dö n z ) and BBM enriched with soil water extract, under 16:8 hi n d á k into the genus Mychonastes, based on their light:dark cycle at 18 °C and 70 µmol.m2.s–1. Morpho- observations from scanning– and transmission– logy was examined using an Olympus BX60 light electron microscopy. Today, the species is microscope with Nomarski’s DIC optics. Fluorescent regarded as a member of the genus Muriella staining with DAPI and Syto11 was carried out (Gu i r y & Gu i r y 2011). In hi n d á k (1982), Muriella following the protocol of za c h l e d e r & ce p á k (1987), aurantiaca vi s c h e r was synonymized with M. and using a Zeiss Axioskop2 for observation. zofingiensis (dö n z ) hi n d á k , but it has since been The genes of interest were the nuclear small demonstrated that M. aurantiaca belongs in a subunit (18S) rRNA gene, which is commonly used separate chlorophycean genus, Pseudomuriella in green algal systematic studies, and the increasingly popular plastid gene encoding the large subunit of (ha n a G a t a 1998; Fu č í k o v á et al. 2011) and that M. zofingiensis is a lineage distinct from Muriella ribulose 1,5–bisphosphate carboxylase/oxygenase (rubisco), rbcL, shown to perform well at the genus– (ka l i n a & punčochářová 1987; ha n a G a t a 1998), and species–level (ri n d i et al. 2007). We used the Mychonastes (kr i e n i t z et al. 2011), and Chlorella 18S gene to determine the relationships of the strains sensu stricto (hu s s et al. 1999). of interest to chlorophycean and trebouxiophycean As part of a larger molecular–phylogenetic genera, in which they had been classified previously: work focused on the genus Bracteacoccus, Bracteacoccus te r e G , Chlorella Be i j e r i n c k , several strains bearing this generic name were Muriella pe t e r s e n , and Mychonastes si M p s o n et va n demonstrated to belong to lineages distinct from va l k e n B u r G . We also included other taxa putatively Bracteacoccus (unpublished data). A previous related to the strains of interest in our analyses: study focused on one of these lineages, the genus e.g., Dictyococcus Ge r n e c k , Planktosphaeria G.M. sM i t h , Pseudomuriella ha n a G a t a , and Radiococcus Pseudomuriella ha n a G a t a , to which two former sc h M i d l e , which were selected based on similarities in Bracteacoccus species [B. engadinensis (ko l et gross morphology. The rbcL data were used for a finer– ch o d a t ) st a r r and B. terrestris (ko l et ch o d a t ) scale analysis focused on the Chlorophyceae. Two st a r r ] were transferred (Fu č í k o v á et al. 2011). other variable markers were used to assess diversity In the present study, we sought to determine within the newly characterized lineage: the nuclear phylogenetic relationships of the putatively ribosomal internal transcribed spacer 2 (ITS2), the misclassified Bracteacoccus cinnabarinus, utility of which for species–level taxonomy is reviewed B. minutus and Muriella zofingiensis, taking in co l e M a n (2003), and the plastid–encoded gene for advantage of the available type cultures. the elongation factor TU (tufA), another promising, Additionally, we examined three UTEX extreme although not–yet widely utilized, species–level marker environment (EE) strains from the Negev Desert (Fa M a et al. 2002). DNA was isolated using the PowerPlant DNA (Israel) – UTEX EE76, EE82, and EE83, all Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, designated as Bracteacoccus sp. – to determine CA, USA). Primers from sh o u p & le w i s (2003) their phylogenetic affiliations. were used for the 18S gene, with PCR conditions as This study serves as an example of a follows: 94 ºC for 1 min, 54 ºC for 45 s, 72 ºC for 1 common problem: unclear relationships among min 45 s, repeated 27 times and followed by a final morphologically poor organisms, their tangled extension at 72 ºC for 8 min 15 s.