evaluating the morphospecies concept in the...

EVALUATING THE MORPHOSPECIES CONCEPT IN THE SELENASTRACEAE(CHLOROPHYCEAE, CHLOROPHYTA)1

Marvin W. Fawley,2 Michelle L. Dean, Stephanie K. Dimmer and Karen P. Fawley

Department of Biological Sciences, North Dakota State University, Fargo, ND 58105, USA

Isolates of the genera Monoraphidium Kom.-Legn., Ankistrodesmus Corda and Raphidocelis Hin-dak emend. Marvan et al. were cultured from twoareas in Minnesota and North Dakota, USA. Theseisolates were identified to species level (when pos-sible), using light microscopy and standard mono-graphs and then characterized by 18S rDNAsequence analysis. Phylogenetic analyses indicatedthat in some cases, 18S rDNA sequences from theseisolates were very similar, but not identical to thesequences of other isolates of the same morphospe-cies from different parts of the world. However,some isolates that were identified as the same spe-cies actually belong to different lineages within theSelenastraceae, whereas other isolates with identicalor nearly identical 18S rDNA sequences possessedrather different morphologies. Overall, our datasuggest that the application of a broad morphospe-cies concept to the Selenastraceae has resulted in anunderestimation of the species diversity of this fam-ily and probably erroneous conclusions about thedistribution of species.

Key index words: Ankistrodesmus; Monoraphidium;morphospecies; Raphidocelis; Selenastraceae

Abbreviations: ANWR, Arrowwood National Wild-life Refuge; ISP, Itasca State Park

The Selenastraceae are ubiquitous freshwater greenalgae with uncertain taxonomy (Krienitz et al. 2001).The commonly observed genera in this family, An-kistrodesmus, Selenastrum, Monoraphidium, and Kirchneri-ella, are generally spindle-shaped, either straight orcurved, and reproduce exclusively by autosporulation.Although genera are delimited by apparently distin-guishable morphological features such as solitary orcolonial habit, presence of mucilage pads, and overallcell shape (Table 1), these features have been shown tobe ambiguous at the genus level by 18S rDNA se-quence analysis (Krienitz et al. 2001). Members of thisfamily are almost always included in phytoplanktonfloras where identification, frequently to species, hasbeen performed by light microscopy. However, the re-sults of Krienitz et al. (2001) bring into question theaccuracy of these identifications.

Recent molecular studies of freshwater green algaehave indicated considerable cryptic diversity amongsome genera and species, most notably Choricystis Fott(Hepperle and Krienitz 2001, Hepperle and Schlegel2002, Fawley et al. 2005) and Chlorella Beijerinck(Huss et al. 1999, Krienitz et al. 2004). These studieshave mostly employed 18S rDNA or rbcL sequenceanalysis. Both of these genes generally evolve too slow-ly to be useful for resolving species-level relationships,which would require examining highly variable re-gions such as the ribosomal ITS (An et al. 1999, vanHannen et al. 2000, Krienitz et al. 2004). Thus, thestudies that have been conducted using 18S rDNA andrbcL have probably revealed only a portion of theactual diversity present.

The only molecular investigation of the diversityamong the Selenastraceae is that of Krienitz et al.(2001). They examined 18S rDNA sequences from11 named species representing the genera Anki-strodesmus, Kirchneriella, Monoraphidium, Podohedriella,and Quadrigula. Their results indicated monophylyfor the Selenastraceae, but the two genera (An-kistrodesmus and Monoraphidium) for which they exam-ined multiple species were both polyphyletic. Nocommonly employed features such as colony forma-tion, cell shape, autospore arrangement, or ultrastruc-ture of the pyrenoids were useful for delimiting thelineages of the Selenastraceae. However, Krienitz et al.(2001) stated that these characters are or may be usefulfor species-level identifications.

The goal of this study was to evaluate the use ofmorphology for species-level identification of mem-bers of the Selenastraceae. For this investigation, weisolated unialgal cultures from sites in Arrowwood Na-tional Wildlife Refuge (ANWR), North Dakota (USA)and Itasca State Park (ISR), Minnesota (USA). Lightmicroscopy-based floras for these two areas have iden-tified numerous species from this family from both lo-cations (Meyer and Brook 1968, Phillips and Fawley2002a,b). Some overlap between the two floras exists atthe species level. By isolating these organisms into cul-ture, we were able to critically compare species iden-tifications by light microscopy with the results ofmolecular analyses using 18S rDNA sequences.

MATERIALS AND METHODS

Site descriptions. ANWR is located in the mixed-grass prai-rie region of east-central North Dakota. Within the refuge,four natural shallow lakes and wetlands on the James River

1Received 19 November 2004. Accepted 26 October 2005.2Author for correspondence: e-mail [email protected].

142

J. Phycol. 42, 142–154 (2005)r 2005 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2006.00169.x

have been stabilized by weirs. Four sites on three lakes, Ar-rowwood Lake, Mud Lake, and Jim Lake, were sampled forthis study. Surface water samples (or just below the ice) werecollected from the ANWR during three consecutive winters,1994–1997 and monthly during 1995.

Samples were also collected from six sites in ISP, Minnesota,including Lake Itasca (mesotrophic), Mary Lake (mesotro-phic), West Twin Lake (moderately dystrophic), North DemingPond (dystrophic), an unnamed dystrophic pond referred to asTower Pond and an unnamed moderately eutrophic pond re-ferred to as Picnic Pond. Phytoplankton and tychoplanktonsamples were taken from these sites once during each seasonfrom fall, 2000 through summer, 2001. Itasca State Park islocated at the transition of the prairie, hardwood forest, andnorthern coniferous forest and is notable as the headwaters ofthe Mississippi River. For more complete descriptions of thesites and their physical and chemical characteristics, see Phillipsand Fawley (2002a, b) and Fawley et al. (2004).

Algal isolations. Algae were isolated using the technique ofPhillips and Fawley (2000) and Fawley et al. (2004). Cultureswere maintained on agar slants and grown in liquid mediawith continuous fluorescent illumination (approximately50–75 mmol photons �m�2 � s�1) at 151C for microscopy andDNA extraction.

Light microscopy and identification. Light microscopy em-ployed a Nikon E600 microscope (Nikon, Melville, NY, USA)equipped with a 60� plan apochromatic objective and dif-ferential interference optics. Digital images were capturedwith a Pixera digital camera (Pixera Corp., Los Gatos, CA,USA). Komarkova-Legnerova (1969), Prescott (1973), Hin-dak (1977), Komarek and Fott (1983), Hindak (1984), Ny-gaard et al. (1986), Hindak (1988), Korshikov (1987) andJohn and Tsarenko (2002), were used for species identifica-tion.

Molecular characterization. Genomic DNA was extractedfrom the isolates, using the technique of Phillips and Fawley(2000) or Fawley and Fawley (2004). The 18S rDNA was am-plified by PCR using the protocol of Fawley and Fawley(2004) with the primer sets NS1 (White et al. 1990) and18L (Hamby et al. 1988), NS1-X and 18L-X (Fawley and Fa-wley 2004) or Med A and Med B (Medlin et al. 1988). The18S rDNA was then sequenced (Fawley et al. 2004) using theprimers NS-1, NS-3, NS-5, NS-7 (White et al. 1990), NS1-Xand 18L-X (Fawley and Fawley 2004), 18G, 18H, 18J, 18L(Hamby et al. 1988), and a new primer, NS3a (5 0 AA-GTCTGGTGCCAGCAGCC 3 0). Sequences were analyzedwith the Staden Package (Staden 1996) and aligned usingGeneDoc version 2.6.000 (Nicholas et al. 1997) and Mac-Clade 4.03 (Maddison and Maddison 2000). Additional pub-lished sequences from the Selenastraceae and representativesof Scenedesmus and Desmodesmus (outgroup taxa) were also

included (Table 2), as well as the sequence from Ourococcusmultiporus, a taxon shown to be allied with the Selenastraceaeby Buchheim et al. (2001). All characters were unambiouslyaligned. The alignment has been submitted to the EMBL-Align database (Lombard et al. 2002) and is also availablefrom the authors. Phylogenetic analyses were performedwith PAUP* 4.0b10 (Swofford 2002). Modeltest 3.5 (Posadaand Crandall 1998) was used to determine the optimal modelof evolution for these data. Maximum likelihood (ML) anal-ysis with the GTRþGþ I model employed rates for the A–Gtransitions of 2.9284 and the C–T transitions of 4.6155. Thesubstition rates for the A–T, G–T, A–C, and G–C transver-sions were 1.0. The gamma distribution for this data set was0.6665 and the proportion of invariable sites was 0.7549.Maximum parsimony (MP) analysis was conducted with thetransition/transversion ratio set from the data using the re-weight function in PAUP. The analysis included 1812 totalcharacters, of which 220 were variable and 115 were parsi-mony informative. Bootstrap analyses were performed with100 repetitions for ML and 1000 repetitions for MP. Pair-wisedifferences among sequences, both number of nucleotidesubstitutions and percent differences, were determined usingPAUP. A neighbor-joining analysis using the total substitu-tions distance matrix was performed with PAUP. This neigh-bor-joining analysis was performed only to graphicallyillustrate the substitution differences among sequences andnot as an inference of phylogeny. As such, bootstrap analysiswas not performed.

RESULTS

Characterization by light microscopy. Seven isolatesfrom ANWR and 11 isolates from ISP identified asSelenastraceae were characterized for this study. TheArrowwood isolate, AS 3-5 (isolated from ArrowwoodLake, 24 February 1995), was identified as Mono-raphidium minutum (Fig. 1). The cells were cylindricaland either arcuate or sigmoid shaped. The cells wererounded at the ends and no pyrenoids were observ-able. Mucilage was not observed. The diameter of thecell helix ranged from 5.8 to 12.2 mm and cell widthranged from 2.4 to 5.6 mm. The fragment of themother cell membrane was unseparated resultingfrom a partial longitudinal rupture.

The Arrowwood isolate MDL 1/12-3 (from MudLake, 12 January 1996) was identified as Raphidocelissubcapitata (formerly referred to Selenastrum carp-ricornutum, see Nygaard et al. 1986) (Fig. 1). The

TABLE 1. Characteristics of the genera included in this study.

Ankistrodesmus Corda—colonial in mucilage with fusiform cells in fasicles (parallel bundles) in which the cells are in contact. Straightor slightly curved, isopolar cells lacking visible pyrenoids.

Kirchneriella Schmidle—cells solitary, with mucilage around each cell. Cells isopolar, arctuate to lunate or spirally twisted, with apyrenoid.

Monoraphidium Komarkova-Legnerova—cells solitary, lacking mucilaginous envelope. Cells fusiform and isopolar, without visiblepyrenoids (naked pyrenoids).

Ourococcus Grobety—cells solitary, without mucilage. Similar to Monoraphidium except possessing an obvious pyrenoid. Sometimesmerged into Keratococcus Pascher.

Podohedriella Hindak—solitary fusiform cells lacking mucilage. Heteropolar, without visible pyrenoid.Quadrigula Printz—colonial in mucilage with cells that lie parallel to one another (as in Ankistrodesmus) but are clearly separate, never

touching. Cells generally with rounded ends; otherwise similar to Ankistrodesmus.Raphidocelis Hindak emend. Marvan et al.—as for Kirchneriella, but without visible pyrenoids. Frequently with incrustations of the cell

wall.

From Marvan et al. (1984), except Podohedriella from Hindak (1988) and Ourococcus from Shubert (2003).

SELENASTRACEAE SPECIES CONCEPT 143

arcuate- or sigmoid-shaped cells were surrounded byconspicuous mucilage. Cell dimensions were 5.6–11.7mm helical diameter and 1.9–3.6mm cell width.The ends of the cells frequently appeared subcapitateas a result of the helical twisting of the cell ends.Autospore production was similar to AS 3-5, with thefragment of the mother cell membrane unseparatedresulting from a partial rupture. In this case, the rup-ture produced an irregular opening not located on thelongitudinal axis.

One ANWR isolate, AN 7-8 (from Arrowwood Lake,July 18, 1995), was identified as M. griffithii (Fig. 1).The cells were straight to nearly straight and fusiform.

The cells came to a gradual point and no pyrenoid wasvisible. The average cell length and width were 36.5and 3.6mm. Cell length ranged from 27.7 to 63.3mm,and cell width ranged from 2.4 to 7.3mm. The frag-ment of the mother cell membrane was separated intotwo halves by an equatorial rupture. This isolate couldalso be compared to M. intermedium Hindak (Hindak1984), which it resembles in the shape of the apices,but the cells of our isolate are larger than for M. inter-medium.

Arrowwood isolate MDL 1/12-5 (from Mud Lake, 12January 1996) was identified as M. pusillum (Fig. 1), butappeared in many ways intermediate between M. con-

FIG. 1. Dendrogram depicting partial results of a neighbor-joining analysis of 18S rDNA sequences from Selenastraceae isolates, andaccompanying light micrographs. Two sections of the dendrogram that included new isolates are shown. The analysis employed adistance matrix constructed using total number of substitutions between sequences and illustrates the total sequence differences. Branchlengths indicate absolute numbers of substitutions among sequences from isolates and published sequences.

MARVIN W. FAWLEY ET AL.144

tortum and M. pusillum. Cells were typically curved orslightly arcuate, as in M. contortum, but only slightly so.This shape is within the range described for M.pusillum. Cells ranged from 13.4 to 32.3mm long � 1.8to 4.9mm wide.

One isolate from ANWR, AS 7-3 (from ArrowwoodLake, 18 July 1995), was identified as M. convolutum(Fig. 1). The cells were arcuate or widely fusiform andthe ends gradually came to a point. The ends of thecells did not lie within one plane and pyrenoids werevisible. The average cell length and width were 7.3 and2.8mm. Cell length ranged from 5.3 to 11.6mm, andcell width ranged from 1.7 to 4.6mm. The fragment ofthe mother cell membrane was unseparated resulting

from a partial longitudinal rupture. Autospores tendedto have sharper ends than that of adult cells.

Two ANWR isolates, AS-11 (from Arrowwood Lake,December 1994) and AS 6–3 (from Arrowwood Lake,29 June 1995), were identified as M. contortum al-though, the isolates were morphologically distinct(Figs. 1 and 2). Cells of AS-11 were slightly arcuate toarcuate, tapered gradually to fine points, with dimen-sions 10.2–27.4mm long � 2.5–5.8mm wide. The endsof the cells were in separate planes. When viewed intwo dimensions, the ends of the cells appeared to near-ly touch, which is not typical for M. contortum. Cells ofAS 6-3 varied greatly from straight to markedlycurved. The cells were narrowly or broadly fusiform

FIG. 2. Dendrogram depicting partial results of a neighbor-joining analysis of 18S rDNA sequences from Selenastraceae isolates, andaccompanying light micrographs. One section of the dendrogram that included new isolates is shown. The analysis employed a distancematrix constructed using total number of substitutions between sequences and illustrates the total sequence differences. Branch lengthsindicate absolute numbers of substitutions among sequences from isolates and published sequences. Monoraphidium sp. Itas 9/21 14-6w,which was resolved as a separate basal lineage, is also shown.


and pointed. Many cells were sigmoid as is typical forM. contortum. No pyrenoid was visible within thechloroplast. Cell length ranged from 11.5 to 35.0mm,and cell width ranged from 1.3 to 6.5mm.

Itasca isolate Pic 8/18 P-7w (from Picnic Pond phyto-plankton, 18 August 2001) was identified as M. pusillum(Fig. 1), but differed somewhat from the ArrowwoodM. pusillum isolate and the description of Komarkova-Legnerova (1969). The Itasca isolate was shorter thanthe Arrowwood isolate, with overall dimensions 11.1–19.4mm � 2.7–4.5mm for adult cells. These dimen-sions are actually more in keeping with the originaldescription of M. pusillum. In addition, autospores ofthe Itasca isolate were much broader than illustratedby Komarkova-Legnerova. However, Komarkova-Leg-nerova indicates that this species is highly variable. Theisolate can also be compared to M. braunii, but the cellsof our isolate are much shorter than those of that spe-cies, and the somewhat finger-like processes at theends of the cell also distinguish our isolate from M.braunii. Isolate Pic 8/18 P-7w has a shape similar to thatof M. neglectum Heynig and Krienitz (1982), but ourisolate is much smaller and the mucilage pads charac-teristic of M. neglectum were never observed. Thus,we place this isolate in the highly variable species,M. pusillum.

Isolate Itas 9/21 S-2w (from Lake Itasca phytoplank-ton, 21 September 2000) was identified as Anki-strodesmus gracilis (Fig. 1). This isolate matched the de-scription of A. gracilis in Komarkova-Legnerova (1969)exactly. Cells were 30 to 35mm � 1.9 to 3.7mm, strong-ly arcuate, and typically occurred in bundles of fourcells.

Isolate Itas 8/18 S-1d (from Lake Itasca phytoplank-ton, 18 August 2001, Fig. 1) could not be placed withconfidence in any species of Komarkova-Legnerova(1969) or Korshikov (1987). Cells were 41–68mm � 2.8–3.7mm, cylindrical or slightly narrowed atthe center, with ends of older cells tapering to finger-like ends. Young cells were more spindle-shaped andoften somewhat sigmoid. This shape most strongly re-sembles M. braunii, except in that species, the cells arebroader than that for our isolate. Monoraphidiumbraunii also has a prominent pyrenoid (Komarkova-Legnerova 1969), a feature not seen in our isolate.

Isolate Itas 9/21 14-1w (from Lake Itasca phyto-plankton, September 21, 2000), Monoraphidium sp.(Fig. 1), somewhat resembles M. braunii and M. obtu-sum (Korsh.) Kom.-Legn., with straight cells with endsof the cells that are clearly rounded. Overall dimen-sions (34 to 52mm � 2.6 to 3.9mm) are within therange of M. braunii. However, the autospores are spi-rally twisted and straighten with age. Autosporangiabecome inflated and produce four or eight autospores.In addition, mucilage is secreted from one end of thecell, a feature not mentioned for either M. braunii orM. obtusum (Komarkova-Legnerova 1969).

Itasca isolate NDem 9/21 T-6w (from North DemingPond tychoplankton, 21 September 2000), Ankistro-desmus sp. (Fig. 2), somewhat resembled Ankistrodesmus

densus Korsh. in colony form, but differed from thedescription of Komarkova-Legnerova (1969) in thatcells were not cylindrical, but rather were constricted atthe midpoint and somewhat shorter than the descrip-tion. Autospores often formed spiral bundles that per-sisted as a colony of adult cells. In other cases, the cellsstayed together in parallel bundles of three or fourcells. Overall cell size ranges from 27 to 55mm � 1.7 to3.5mm.

Isolate Itas 8/18 M-7w (from Lake Itasca phyto-plankton, 18 August 2001) strongly resembled Anki-strodesmus fusiformis (Fig. 2). Cells were 42–56mm �2.5–3.7 mm and generally straight or nearly so. Cellsoften remained together in groups of 2 to 4,either in parallel bundles or irregularly crossed. Ourisolate differs from typical A. fusiformis in that many ofthe colonies were present as parallel bundles, whereasin typical A. fusiformis, the cells of colonies are crossed(Komarkova-Legnerova 1969, Korshikov 1987). In theformation of parallel bundles, our isolate resemblesA. falcatus (Corda) Ralfs, which has been consideredconspecific with A. fusiformis by Hindak (1988) due tovariability in colony form.

Isolate NDem 8/18 T-6d (from North Deming Pondtychoplankton, 18 August 2001) also resembledA. fusiformis in some respects (Fig. 2). Cells were35–40mm � 2.6–4.1mm, nearly linear to somewhatspirally twisted. A distinguishing feature of this isolatewas the production of mucilage from one pole of manyof the cells, which often gave the appearance of a ca-pitate end similar to that of the genus PodohedraDuringer. In addition, no cruciate colonies typical forA. fusiformis were observed. In fact, organized coloniesof any type were infrequent, although some parallelbundles of cells were observed. Many ‘‘colonies’’ weresimple loosely connected cells, often in linear series orsomewhat so. These characteristics do not match any ofthe described species of Ankistrodesmus.

Mary 8/18 T-2w was isolated from a tychoplanktonsample from Mary Lake in ISP, 18 August 2001.Most cells were in parallel bundles of generally fourcells, similar to A. falcatus (Corda) Ralfs (Fig. 2). Occa-sionally, cells were spirally twisted and in groups offour cells twisted together. A few group of cells formedirregular cruciate patterns, similar to A. fusiformis. Thisisolate was re-plated and individual colonies werechecked to see if multiple taxa were mixed togetherin the original isolate, but all re-isolates possessed thethree colony morphologies. Individual cells were quitethin relative to their length, with dimensions 40–56mm � 1.7–3.1mm, and nearly straight or slightly ar-cuate to somewhat helical. This isolate fit the descrip-tion of A. falcatus, although the presence of the spirallytwisted colonies, never mentioned by Komarkova-Leg-nerova for this species, makes this identification ques-tionable. The isolate could also be identified as A.stipitatus by the criteria of Komarkova-Legnerova(1969), but the cells are not of the same dimensionsand that taxon also lacks the diversity of colonyform.


Isolates Mary 9/21 T-5w (from Mary Lake tycho-plankton, 21 September 2001) and NDem 9/21 T-9d(from North Deming Pond tychoplankton, 21 Septem-ber 2001) matched almost perfectly the description ofM. saxatile Komarkova-Legnerova (Fig. 2). Cells di-mensions were 26–37mm � 2.4–3.8 mm. Older cellswere linear and younger cells tended to be arcuate,but never sigmoid. In some cells, a small pyrenoid wasvisible. Cells were typically attached to each other atone end by a conspicuous pad of mucilage; mucilagerarely was apparent at both ends of a cell. All of thesefeatures are consistent with M. saxatile, as is the tycho-planktonic habitat.

Cells of isolate Itas 9/21 14-6w (from Lake Itascaphytoplankton, 21 September 2000) were very longand thin, with dimensions 70–88mm � 2.4–5mm (Fig.2). The cells become somewhat inflated before auto-spore production. Cells were typically straight with theends drawn out to fine points; however, autospores justreleased from the mother cell sometimes were some-what bent or twisted because the two parts of themother cell wall did not completely separate. Inmany cases, cells remained attached in a semi-linearsequence. In shape and dimensions, this isolate resem-bles M. komarkovae Nygaard (M. setiforme (Nygaard)Komarkova-Legnerova in Komarkova-Legnerova1969). However, in M. komarkovae, the mother cell

wall cleanly separates into two parts, which is not thecase for our isolate. Another species, A. acicularis (A.Braun) Korsh., which Komarkova-Legnerova reducedto synonomy with M. griffithii, is similar in shape andsize to our isolate. However, once again the separationof the mother cell wall in that species is into two parts,which is not the case for our isolate. Therefore, thisisolate cannot be referred to any named species.

Analyses of 18S rDNA sequence data. Eighteen 18SrDNA sequences were generated from the isolatesexamined. Table 2 indicates the GenBank accessionnumbers of these new sequences. The pair-wise dif-ferences in nucleotide sequences (excluding introns)are shown in Table 3. Mary 9/21 T-5w and NDem 9/21 T-9d, both identified as M. saxatile, possessed iden-tical 18S rDNA coding sequence, but Mary 9/21 T-5wpossessed putative Group 1 introns that were lackingin NDem 9/21 T-9d. Itas 8/18 M-7w, A. fusiformis, andMary 8/18 T-2w, A. sp., also produced identical se-quences. The maximum variation among18S rDNAcoding regions observed among the Selenastraceaewas 3% between the sequence from Arrowwood iso-late AS 7-3, identified as M. convolutum, and the pub-lished sequence for Quadrigula closterioides.

Phylogenetic analyses of the new Selenastraceae se-quences plus the sequences included in the analyses ofKrienitz et al. (2001) and a set of sequences from the

TABLE 2. GenBank accession numbers of 18S rDNA sequences used in this study.

Taxon GenBank accession number

Ankistrodesmus bibraianus (Reinsch) Kors. Y16938Ankistrodesmus fusiformis Corda Itas 8/18 M-7w AY846370Ankistrodesmus fusiformis X97352*Ankistrodesmus gracilis (Reinsch) Korsh. Itas 9/21 S-2w AY846371Ankistrodesmus gracilis Y16937*

Ankistrodesmus sp. Mary 8/18 T-2w AY846372Ankistrodesmus sp. NDem 8/18 T-6d AY846373Ankistrodesmus sp. NDem 9/21 T-6w AY846374*Ankistrodesmus stipitatus (Chod.) Kom.-Legn. X56100Kirchneriella aperta Teiling AJ271859*

Monoraphidium braunii (Nag. in Kutz.) Kom.-Legn. AJ300527Monoraphidium contortum (Thur.) Kom.-Legn. AS-11 AY846375*Monoraphidium pusillum MDL 1/12-5 AY846376Monoraphidium convolutum (Corda) Kom.-Legn. AS7-3 AY846377Monoraphidium dybowskii; (Wolosz.) Hindak et Kom.-Legn. Y16939Monoraphidium griffithii (Berk.) Kom.-Legn. AN7-8 AY846378Monoraphidium minutum (Nag.) Kom.-Legn. AS3-5 AY846380Monoraphidium neglectum Heynig et Krienitz AJ300526Monoraphidium contortum (Printz) Kom.-Legn. AS6-3 AY846382Monoraphidium pusillum Pic 8/18 P-7w AY846383Monoraphidium saxatile Kom.-Legn. Mary 9/21 T-5w AY846384*Monoraphidium saxatile NDem 9/21 T-9d AY846385Monoraphidium sp. Itas 8/18 S-1d AY846386Monoraphidium sp. Itas 9/2114-1w AY846387Monoraphidium sp. Itas 9/21 14-6w AY846379Monoraphidium terrestre (West et G.S. West) Kom.-Legn. Y17817*

Ourococcus multisporus Bischoff et Bold AF277648Podohedriella falcata (Duringer) Hindak X91263Quadrigula closterioides (Bohlin) Printz Y17924*Raphidocelis subcapitata (Korsh) Nygaard et al. MDL 1/12-3 AY846381*Raphidocelis subcapitata AF169628*

Desmodesmus communis (Hegew.) Hegew. X73994Scenedesmus obliquus (Turp.) Kutz. X56103

Bold font indicates sequences generated in this study. Asterisks indicate sequences with 1 or more putative introns.


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).


Chlorophyceae (results not shown) resulted in strongsupport for monophyly of the Selenastraceae, as al-ready determined by Krienitz et al. (2001). Subse-quent analyses utilized the apparent closest relativesof the Selenastraceae, Scenedesmus and Desmodesmus, asoutgroup taxa. Portions of the results of a neighbor-joining analysis of these sequences are depicted in Figs.1 and 2. The analysis depicts the actual substitutiondifferences among the sequences from our isolates andthe most similar published sequences. This analysis wasintended to show the diversity among our isolates anddoes not represent a phylogenetic analysis. Thus, nobootstrap values are shown.

The results of the ML analysis are depicted in Fig. 3,with bootstrap values for both the ML analysis and theMP analysis shown. The results show excellent boot-strap support (100%) for the monophyly of theSelenastraceae, but there is little support for internalnodes. Bootstrap support greater than 70% was onlyachieved for a few terminal lineages. The low bootstrapsupport is probably due to the relatively small numberof variable characters employed in the analyses.

Among the Selenastraceae sequences, there wereonly 87 parsimony informative characters. Our results,like those of Krienitz et al. (2001), show no support fortraditional generic concepts within the Selenastraceae.

More important for this study than the relationshipsamong different lineages of the Selenastraceae is thecomparison of molecular and morphological data.Some of our isolates, such as A. fusiformis Itas 8/18 M-7w, A. gracilis Itas 9/21 S-2w and Raphidocelis subcapitataMDL 1/12-3, produced 18S rDNA sequences very sim-ilar to the published sequences for the same taxa (Table3, Fig. 1). In some cases, we analyzed multiple isolatesthat were referable to the same species, including M.pusillum, M. contortum, M. minutum and M. saxatile. Our2 isolates of M. saxatile, Mary 9/21 T-5w and NDem 9/21 T-9d, share identical coding sequences, althoughthey differ in intron content. The sequences from the 2isolates identified as M. pusillum (MDL 1/12-5 and Pic8/18 P-7w), however, differed by 36 substitutions (Ta-ble 3, Fig. 1) and were resolved in different lineages(Fig. 3). The sequence from M. pusillum MDL 1/12-5actually was identical to that from isolate AS6-3, iden-tified as M. contortum, (Table 3, Fig. 1). The sequence ofPic 9/19 P-7w, identified as M. sp., differed from M.braunii (AJ300527) by only 7 substitutions (Table 3, Fig.1) and this isolate did possess some features similar toM. braunii. Sequences from the 2 isolates identified asM. contortum (AS6-3 and AS-11) differed by 38 substi-tutions (Table 3, Fig. 1) and were also resolved in dif-ferent lineages (Fig. 3).

The additional unidentified isolates Mary 8/18 T-2w,NDem 8/18 T-6d, and NDem 9/21 T-6w were all re-solved as sister taxa to A. fusiformis (X97352) and A.stipitatus (X56100) (Fig. 3). In fact, Mary 8/18 T-2wproduced a 18S rDNA sequence identical to the se-quence of the Itasca A. fusiformis isolate, Itas 8/18 M-7w(Table 3, Fig. 2).

Sequences from the isolates Itas 9/21 14-1w and Itas8/18 S-1d differed from each other by only a singlesubstitution and from the sequence of AS-11, identi-fied as M. contortum, by 4 and 5 substitutions, respec-tively (Table 3, Fig. 1). All of these isolates, plus M.griffithii AN7-8, were resolved in a lineage that includ-ed M. braunii (Fig. 3). The final isolate examined in thisstudy, Itas 9/21 14-6w (Fig. 2), which could not beidentified to species, was resolved without significantbootstrap support as a basal taxon of the Selena-straceae (Fig. 3).

DISCUSSION

Morphological analysis by light microscopy of our18 isolates from the Selenastraceae indicated that 12 ofthe isolates could be tentatively identified as speciesincluded in current monographs. Two isolates wereidentified as Monoraphidium saxatile, 2 as M. contortum, 2as M. pusillum and one isolate each as Ankistrodesmusfusiformis, A. gracilis, M. convolutum, M. minutum, M.griffithii, and Raphidocelis subcapitata. Of these identifi-

FIG. 3. Phylogram resulting from a maximum likelihoodanalysis of 18S rDNA sequences from the Selenastraceae, andincluding Desmodesmus communis and Scendesmus obliquus as out-groups. Bootstrap values for maximum likelihood are shown inbold; values for maximum parsimony are shown in plain text.Only values greater than 70% are given.


cations, only the isolates referred to M. saxatile, A.fusiformis, A. gracilis and R. subcapitata fit the publisheddescriptions without exception. In other cases, therewere differences between our isolates and publisheddescriptions, but these differences may have been un-detectable without culturing. The multiple isolatesidentified as M. contortum and M. pusillum differedfrom each other, but still fit within the broad defini-tions of the species.

The named isolates from ANWR, with the excep-tion of R. subcapitata, had been found previously fromANWR, with identifications based upon light micros-copy from preserved natural samples (Phillips and Fa-wley 2002a,b). During periods of ice cover, M. minutumwas reported as a frequently dominant species, M. pus-illum, M. contortum, and M. convolutum were very fre-quent, and M. griffithii was only occasional found.Although R. subcapitata was not found in the previousstudy, it is very likely that specimens of that specieswould have been referred to M. minutum, which hasvery similar cell morphology but lacks mucilage. Themucilage would have been very difficult to see with thecounting method employed. Additional species foundfrom ANWR by light microscopy but not observed inthis study included 4 species of Ankistrodesmus, only 1 ofwhich, A. bibraianus (Reinsch) Korshikov, was common-ly found.

Previous work on the Itasca State Park algal flora(Meyer and Brook 1968) indicated several species ofSelenastraceae that were not detected in this study, in-cluding A. falcatus (Corda) Ralfs, A. falcatus var. tumidus(West et West) G. S. West, A. spirilis (Turner) Lemm.,M. griffithii (as A. falcatus var. acicularis (Braun) G. S.West and 3 species of Kirchneriella. One of our isolates,Itas 9/21 14-6w, identified simply as Monoraphidium sp.,was very similar to the description of A. acicularis, (seeresults) which was a later combination of A. falcatus var.acicularis (Korshikov 1987). Itas 9/21 14-6w may rep-resent the taxon that Meyer and Brook (1968) referredto A. falcatus var. acicularis. Of the isolates that we iden-tified from ISP, both A. gracile (as Selenastrum westiiSmith) and M. saxatile (as A. falcatus var. stipitatus(Chod.) Lemm.) had previously been detected in thepark. We were unable to assign additional isolates fromISP to any named species, with any confidence.We are also confident that additional taxa fromthe Selenastraceae are present in ISP. We have com-monly observed Quadrigula sp. and additional An-kistrodesmus spp. among tychoplankton samples, buthave not attempted species identification from fieldmaterial.

Although M. spiralis and M. griffithii have both beenreported from ISP and ANWR, we did not culture iso-lates from these 2 species from both sites. Otherwise,there is no reported overlap of the Selenastraceae fromthese locations. We did not also find any additionaltaxa that were presented at both sites in this study. Thecoccoid green algal communities of these locationshave also been found to be largely distinct by analysesof 18S rDNA (Fawley et al. 2004).

Analysis of 18S rDNA sequences from our isolatesand previously published sequences from the Se-lenastraceae indicate the monophyly of the family,but provide little internal resolution. Greater resolu-tion would probably be achieved using more highlyvariable sequences such as rbcL or ribosomal ITS.However, analyses of 18S rDNA sequences allow anassessment of our morphology-based species identifi-cations. The comparison of the relationships amongour isolates based upon sequence analysis and the re-sults of our morphological analysis indicate only partialconcordance, with some striking contradictions. Ourisolate identified as A. fusiformis differed from the pub-lished sequence for an A. fusiformis, SAG 2005 isolatedfrom Germany, by only a single nucleotide. However,we also had additional isolates that differed somewhatfrom A. fusiformis in morphology, but that produced18S rDNA sequences either identical or nearly identi-cal to our A. fusiformis isolate. Another of our isolates,Itas 9/21 S-2w, was an excellent fit for the descriptionof A. gracilis, and the 18S rDNA sequence of this isolatediffered from the published sequence for an A. gracilisisolate from England, SAG 278-2, by five nucleotidesubstitutions. The isolate MDL 1/12-3, identified asRaphidocelis subcapitata, differed by only four substitu-tions from the published sequence for R. subcapitata,that was produced from an isolate, UTEX 1648, from aNorwegian lake. Our 2 isolates of M. saxatile, a speciesthat has not been previously examined by molecularmethods, produced identical 18S rDNA coding se-quences, but differed in intron content. Although se-quences from some of our isolates were very similar topublished sequences from European isolates, in nocase, the sequences were identical.

In other cases, the correspondence between mor-phological and molecular results was not as clear. Wehad 2 isolates, MDL 1/12-5 and Pic 8/18 P-7w, thatwere identified as M. pusillum, although with some res-ervations for both isolates. The 18S rDNA sequences ofthese 2 isolates differed by 36 substitutions and theywere resolved in separate lineages of the Selena-straceae. On the other hand, M. pusillum isolate MDL1/12-5 and isolate AS6-3, identified as M. contortum,produced identical 18S rDNA sequences. An addition-al isolate identified as M. contortum, AS-11, was also re-solved in a separate lineage from AS6-3 by molecularanalyses. Finally, the sequences generated from isolatesItas 8/18 S-1d, Itas 9/21 14-1w and AS-11 differedfrom each other by a maximum of five substitutions,but the morphology of AS-11 was dramatically differ-ent from the other two isolates. Isolate AS-11 wasstrongly curved and identified as A. contortum(although, not a perfect match), whereas the other 2isolates were almost perfectly straight.

In several cases, the characters used to identify spe-cies were inconsistent with the molecular results. Notonly did isolates referred to as the same species fall intodifferent lineages, but also isolates with very similar18S rDNA sequences occasionally possessed morpho-logies that would, without question, result in different


species identification. However, the isolates that weidentified as the same species using current keys thatpossessed somewhat different morphologies were al-ways placed in separate lineages by molecular analyses.For example, AS-11 and AS6-3 could both be con-sidered M. contortum based on cell size, shape, and thegradually pointed cell ends. However, the cells certain-ly were not exactly the same and represent oppositeextremes of the variability attributed to this species byKomarkova-Legnerova (1969).

There is, or course, the possibility that analysesbased only on the single gene, 18S rDNA, may be mis-leading. However, the conservative nature of the 18SrDNA makes this rather unlikely. It is more likely thatthere is even more variability among our isolates thanthat is revealed by these sequences. Additional studiesutilizing a multigene approach should be conducted toenhance these results.

Our results indicate that the characters used toidentify species within the Selenastraceae are not, infact, defining species-level lineages. The charactersconsidered by Komarkova-Legnerova (1969) to be‘‘good interspecific features’’ include: ‘‘(a) Shape ofadult cells, (b) intensity of curvature . . ., (c) shape ofthe colonies with respect to the fascicles, (d) presenceof a mucous layer and its consistence, (e) presence of apyrenoid, (f) range of dimensions, and (g) terminationof the cells.’’ With this array of characters, one can, infact, differentiate among our several isolates, includingthose that we considered to be conspecific based onmorphology. However, these features are clearly notconsistent within any specific lineage and do not pro-vide clear information on the evolutionary relation-ships among isolates.

Our results can be interpreted in several ways. Insome cases, one could argue that all of the organisms ina well-supported lineage represent a particular speciesand that the previous species definition was, in fact, notbroad enough. For example, there is little morpholog-ical variation in cell shape among the well-supportedlineage depicted in Fig. 2 (excluding the 2 M. saxatileisolates) that includes the previously published se-quences from A. fusiformis and A. stipitatus as well as 4of our isolates from ISP. These sequences vary by amaximum of 11 substitutions. The differences betweenthe published descriptions of A. fusiformis and A.stipitatus are primarily length (A. stipitatus is longer)and colony form (A. stipitatus forms fasicles and Afusiformis cruciate or irregular aggregates). These fea-tures were also variable among our isolates from thislineage, in some cases, preventing us from placing aspecies name on isolates. Hindak (1988) also recog-nized the variability of the colony form, arguing thatA. fusiformis and A. falcatus (Corda) Ralfs should beconsidered conspecific.

The well-supported lineage including M. braunii aswell as our isolates, identified as M. pusillum (Pic 8/18 P-7w) and M. griffithii (AN7-8), vary by a maximum of 10substitutions, or approximately the same variation asthe A. fusiformis/A. stipitatus lineage. However, members

of this lineage vary considerably in cell size and theshapes of the ends of the cells. It is unlikely that anyonedescribing taxa from morphological data would com-bine these isolates in a single taxon.

Even less divergent (based on sequence data) thanthe M. braunii lineage is the lineage comprising ourisolates identified as M. contortum (AS-11), M. sp. (Itas9/21 14-1w) and M. sp. (Itas 8/18 S-1d). The sequencesfor these isolates vary by a maximum of 5 substitutions,but the cells of AS-11 are strongly curved whereasthose of the other 2 isolates are straight or very nearlyso. Curvature is one of the primary characters used todelineate species of Monoraphidium. Thus, these isolateswould never be considered the same species by mor-phological analysis.

Finally, two isolates, MDL 1/12-5 and AS6-3, pos-sessed identical 18S rDNA coding sequences, but themorphologies varied significantly. The isolate MDL1/12-5 was identified (with reservations) as M. pusillum.The cells were nearly straight to moderately lunate,and occasionally the ends were very slightly sigmoid.AS6-3 was identified with confidence as M. contortum,and the cells were highly recurved and sigmoid. Theseexamples make it difficult to accept the idea that spe-cies need to be more broadly defined while retainingthe same diacritic characters.

Another possible explanation for our observations isthat species are too broadly defined by the morpho-logical characters that are presently used. Thus, thevariation among isolates that we observe at the molec-ular level is not concordant with the morphologicalspecies definitions. If this explanation is accurate, wewould expect those isolates that matched the most nar-rowly defined species to be the best matches at themolecular level. The most narrowly defined taxaexamined in this study were probably A. fusiformis, A.gracilis, and A. saxatile, which were defined by both celland colony morphologies. Our isolates that matchedthe descriptions of A. fusiformis and A. gracilis did, infact, produce 18S rDNA sequences that were quite sim-ilar to the published sequences for these species. How-ever, the isolates identified as A. fusiformis were notmonophyletic in our analyses, although 18S rDNA isnot variable enough to be confident in this assessment.

Thus, we are left with the explanation that the di-acritical characters used to delimit species in theSelenastraceae, at least for the polyphyletic generaAnkistrodesmus and Monoraphidium, are not reliable.Our results show examples of isolates with identical18S rDNA sequences but morphological variation, andisolates with very different sequences but similarmorphologies. The best explanation for the patternsof our results is that species are currently defined toobroadly under the morphological concept. This resultis not surprising. Some monographs on the Se-lenastraceae have mentioned high levels of variabilityamong isolates referred to a single taxon, with the sug-gestion that this variability may actually represent mul-tiple taxa (i.e. Komarkova-Legnerova 1969, Nygaardet al. 1986).


Our results also indicate that it is very unlikely that aresearcher would be able to accurately identify all Se-lenastraceae from natural samples. For our analysis, weessentially forced isolates into existing species defini-tions (based mostly on European isolates) where thatwas possible. However, we were unable to match sev-eral isolates with any named species. When examininga natural sample, it is often impossible to tell whetherthe range of variation observed represents one or mul-tiple taxa, and frequently autospore production cannotbe observed. However, the general tendency is to placea name on an organism when it falls within the broadtaxon description. By considering that our results, inaddition to those of Krienitz et al. (2001), suggest thatboth existing genera and species in the Selenastraceaeare not well defined by existing criteria, then it is es-sentially impossible to accurately place a species nameon a member of the Selenastraceae from a naturalsample, using only characters visible with light micros-copy.

Our results and those of Krienitz et al. (2001) pointto the need for a massive effort to understand the di-versity of the Selenastraceae and determine reliablecharacters for identification. This effort should employmultiple genes that are more variable than 18S rDNAin concert with morphological analyses. These organ-isms are so frequently encountered in water samplesthat it is, indeed, amazing that we have so little under-standing of what constitutes a species and how to iden-tify taxa at any level. The number of isolates that wecould not assign to any described species as well as ourobservations of the variability of these organisms innatural samples lead us to speculate that there may behundreds of taxa within the family world-wide. Ourconclusions have far-reaching significance. Our resultsindicate that many of the diacritical characters used toidentify species are not reliable. Our results alsostrongly suggest that the species-level diversity of theSelenastraceae has been greatly underestimated. As aresult, species identified using existing monographshave probably been misidentified in many cases. Be-cause our concept of species has been flawed, we reallyhave very little understanding of the true distributionand ecology of any species of Selenastraceae.

We thank Matt Hoffman and Joni Johnson for technical as-sistance and Phillip McClean and Rian Lee for the use of theautomated sequencer. We thank two anonymous reviewerswho provided excellent comments that resulted in substantialimprovements to the manuscript. This material is based uponwork supported by the National Science Foundation underGrant Nos. DBI-0070387, DEB-0128952 and MCB-0084188.Additional support was provided by the North Dakota WaterResources Research Institute.

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