Phylogenetic position of Jaoa, a green algal genus endemic to China

Phylogenetic position of Jaoa, a green algal genus endemicto ChinaHuan Zhu,1,2 Shuang Xia,1,2 Qi Zhang,1,2 Guo-Xiang Liu1* and Zheng-Yu Hu1

1Key Laboratory of Algal Biology, Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan, and 2Universityof Chinese Academy of Science, Beijing, China

SUMMARYJaoa prasina, a freshwater green alga endemic to China,was collected from a stream in Hubei province, China.Unialgal cultivation, morphological observation, andphylogenetic analyses of small subunit ribosomal DNAand RuBisCO large subunit sequences were performed.When cultured on agar medium, the alga was irregularlyfilamentous, similar to marine species of Acrochaete.Aplanospores were observed on solid medium. Avesicular-like thallus without rhizoids developed inliquid medium, similar to specimen development innatural habitats. Molecular phylogenetic analysesrevealed that Jaoa was closely related to the marinegenera Acrochaete Pringsheim and Ulvella Crouan &Crouan. The results suggested the genus Jaoa is amember of the family Ulvellaceae (Ulvophyceae), whichcontains mostly marine algae. The family nameJaoaceae should be abandoned. We speculate that Jaoamay have evolved from a marine Ulvellaceae ancestor.

Key words: Acrochaete, Jaoa, phylogeny, Ulvellaceae,Ulvophyceae.

INTRODUCTION

The green algal family Jaoaceae (Chlorophyta) contain-ing a single genus with two species, Jaoa prasina (Jao)Fan and Jaoa bullata (Jao) Fan, has only been docu-mented in China (Jao 1941; Wang et al. 1988; Li & Hu2003; Shi et al. 2004). Jaoa Fan has a wide distribu-tion from Xinglong County, Hebei province, northernChina to Menglun County, Yunnan province, southernChina (Jao 1941; Wang et al. 1988; Li & Hu 2003; Shiet al. 2003, 2004). The young fronds of Jaoa arespherical or ellipsoidal, grass-green or olive in color,with a surface being more or less uneven; the matureplants are disc-shaped, vesicular, olive or dark-green,hollow with stout folds on the surface, and attached tosubstrata by rhizoids (Jao 1941). The thalli of Jaoaclearly have a distromatic (Jaoa prasina) or trilaminar(Jaoa bullata) structure, which is the traditionalspecies-level delimiter in the genus. Cells coalesce toproduce pseudoparenchymatous masses. The globular

inner cells are larger than the outer cells, which areellipsoidal and flask-shaped in outline. Often severalouter cells are attached to an inner cell in the pseu-doparenchymatous masses (Li & Bi 1998).

Viewpoints on the taxonomic position of Jaoa in theChlorophyta have been changing. Jaoa was first identi-fied by Jao in Chongqing in 1941 and established as anew family, Coelodiscaceae, comprising a single newgenus, Coelodiscus Jao (Jao 1941). In 1964 Fanchanged the genus name from Coelodiscus to Jaoabecause Coelodiscus Jao 1941 was a later synonym ofCoelodiscus Baillon 1858, a flowering plant genus(Euphorbiaceae), and hence illegitimate according tothe rules of the International Code of Botanical Nomen-clature. Similarly, the name Coelodiscaceae waschanged to Jaoaceae. The family was placed in theUlotrichales (Fan 1964). Li and Bi (1998) placed thefamily in Chaetophorales in Flora Algarum SinicarumAquae Dulcis. Both Printz (1964) and Silva (1982)considered Jaoaceae lacking in wide acceptance, andmoreover argued that Jaoa prasina and Jaoa bullatawere not distinct species. Printz (1964) classified Jaoa(Jao) Fan in the Chaetophoraceae. Wang et al. (1988),and Li and Hu (2003) observed the ultrastructure ofJaoa, and the former reported that the ultrastructure ofthe chloroplast of Jaoa was similar to members of theUlvaceae. Overall, the diversity and species-leveldelimitation of this genus has been surrounded by con-siderable uncertainty. The predominant focus on mor-phology in previous taxonomic studies has contributedto the uncertainty.

We examined Jaoa obtained from Xianning County,Hubei province (China), in March and identified it asJaoa prasina. We observed the vegetative morphologyboth in natural habitat and in laboratory culture condi-tions, and also reassessed the taxonomic position basedon phylogenetic analyses of small subunit ribosomalDNA (SSU rDNA) and RuBisCO large subunit (rbcL)sequences.

*To whom correspondence should be addressed.Email: [email protected] editor: O. De Clerck.Received 10 August 2012; accepted 1 April 2013.doi: 10.1111/pre.12021

Phycological Research 2013

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© 2013 Japanese Society of Phycology

MATERIALS AND METHODS

Collection and culture

The specimens were collected from a stream below theNanchuan Reservoir (29°42′N, 114°24′E), XianningCounty, Hubei province, China, in March 2011 (Fig. 1).The stream flows slowly, has a depth up to 0.5–0.7 m,and has many stones at the bottom. Specimens andnatural water samples were collected in clean polyeth-ylene bottles and brought to the laboratory on the sameday. Plants were rinsed clean with tap water to removeepiphytes and sediment. The scrubbed plants were cutinto small pieces under a stereoscope (KL1500 LCD;Zeiss, Göttingen, Germany), and a dozen of pieces wereinoculated on BG-11 medium with 1.1% agar in plasticPetri dishes (Allen 1968). The living green algal fila-ments were selected every 2 weeks for incubation onfresh medium until unialgal cultures were obtained.The algal filaments were also cultured in flasks withliquid algal medium (BG-11). Cultures were maintainedat 18°C, 50 mmol m-2 s-1 illumination intensity with a16 : 8 h light : dark (LD) cycle. A culture strain wasdeposited in the Freshwater Algae Culture Collection,Institute of Hydrobiology, the Chinese Academy of Sci-ences (FACHB) under the accession No. FACHB-1441.

Morphological observation

Fresh specimens collected from the stream were sec-tioned under a stereoscope (KL1500 LCD; Zeiss) andfilaments of Jaoa cultured on solid medium were pickedunder the same stereoscope. Morphological traits andmicrostructure were observed using a microscope(DM5000B; Leica, Wetzlar, Germany). Micrographswere taken with a Leica DFC320 digital camera.

DNA extraction and amplification

Total genomic DNA was extracted from fresh thalli fol-lowing the procedures outlined in Hayden and Waaland(2002). Double-stranded amplification of nuclear-encoded SSU rDNA and rbcL genes was performed in atotal volume of 50 mL including 2 mL template DNA(10 ng mL), 1 mM forward primer, 1 mM reverse primer,0.4 mM dNTPs, 5 mM MgCl2, and 1.25 U Taq DNApolymerase. The universal primers designed by Medlinet al. (1988) were used to amplify SSU rDNAsequence. The primers rbcLB and rbcLQ were used toamplify the rbcL sequence following the protocol ofRindi et al. (2008). The SSU rDNA sequence amplifi-cation profile included an initial denaturation at 95°Cfor 2 min, followed by 50 s at 94°C, 50 s at 56°C, 90 sat 72°C for 31 cycles, and a final extension of 7 min at72°C. The rbcL sequence amplification profile con-sisted of a preliminary 1 min denaturing at 95°C, 33cycles of 50 s denaturing at 94°C, 50 s annealing at52°C, 75 s extension at 72°C, and a final extension of5 min at 72°C.

The amplification products were separated alongwith a sample of the control and a 100 bp DNA sizemarker (Promega, Madison, WI, USA) in 1.0% (w/v)agarose gels cast in Tris-acetate buffer (0.04 MTris pH 8.0, 1 mM ethylenediaminetetraacetic acidpH 8.0). The gels were electrophoresed at 100 V for35 min and viewed under ultraviolet light after stainingfor 15 min with ethidium bromide (2 mg mL-1).Excised products were purified with a Qiaquick GelExtraction kit (Qiagen Duesseldorf, Germany) accordingto the manufacturer’s instructions. The products ofpurification were preserved at 4°C.

The purified products were run on an ABI 3700sequencer (Applied Biosystems, Foster City, CA, USA).Sequences were deposited in GenBank under accessionnumbers JN102133 and JN102134.

Phylogenetic analyses

Twenty-four SSU rDNA sequences and 15 rbcLsequences, selected based on a BLAST search orrepresentation of reference species in the relevanttaxonomic class, were downloaded from GenBank(http://www.ncbi.nlm.nih.gov/) and included in the

Fig. 1. Distribution of Jaoa Fan in China. Map supplied by the

State Bureau of Surveying and Mapping, available online at http://

www.sbsm.gov.cn/article/zxbs/dtfw. (•) indicates various provincial

capital cities and (�) indicates national capital city.


2 H. Zhu et al.

alignments. Sequences were aligned using Clustal Xv2.0 (Larkin et al. 2007), and further manually editedand adjusted by eye. Positions of SSU rDNA and rbcLsequences that could not be aligned with confidencewere removed prior to analyses. Sequence alignmentswere exported as NEXUS files from Clustal X and ana-lyzed using neighbor joining (NJ), maximum parsimony(MP), and maximum likelihood (ML) implemented inPAUP* v. 4.0b10 (Swofford 2003).

Trentepohlia aurea and Cladophora albida wereused as outgroup in the SSU phylogeny. In the rbcLphylogeny, two sequences from the genus Trentepohliawere used as outgroup. All characters and characterstate changes were weighted equally and gaps werecoded as missing data in the ML analyses. Modelparameters for ML analyses were determined fromhierarchical likelihood ratio tests using Modeltestv.3.06 (Posada & Crandall 1998). The best-fit models(TrNef + I + G model for SSU rDNA sequences analy-sis and GTR + G model for rbcL sequences) wereapplied to construct a ML phylogenetic tree. Heuristicsearches consisted of 10 replicate searches with ran-domized taxon addition, tree bisection-reconnection,MulTrees, and steepest descent options in effect(Swofford 2003). The same datasets were subjectedto MP analysis. Parameters including base frequen-cies, transition/transversion ratio, proportion of invari-able sites, and site-to-site rate heterogeneityestimated under Modeltest were also used for dis-tance analysis using the NJ method. For all analyses,relative support for branches and stability of treetopology were evaluated by bootstrap analysis inwhich the data were resampled 5000, 1000, and 500times for NJ, MP, and ML, respectively. Bootstrapvalues are presented on the nodes.

RESULTS

Morphological observation

Jaoa prasina (Jao) Fan, Acta Phytotax. Sinica, 9 (1):101, 1964.

Synonyms: Coelodiscus prasina Jao, Studies on thefresh-water algae of China IX. Coelodiscaceae. Sinensia1941, 12:291–298; Jaoa bullata (Jao) Fan, ActaPhytatax Sinica, 9(1): 101, 1964; Coelodiscus bullataJao, Bot. Bull. Acad. Sinica, 1: 255–256, f.1, 1947;Coelodiscus prasinus var. bullatus (Jao) Printz, Hydro-biol., 24:287–288, t.90, f.1–2, 1964.

Morphological description: Thalli were disc-shapedor vesicle-like and grass-green or olive in color. Youngthalli were ellipsoidal or subspherical, with a roughwrinkled surface; mature thalli were disc-shaped andvesicular, with many unequal size folds on the surface.Plants were occasionally solitary but predominatelyclustered in groups on stones at the bottom of the

stream. The diameter of young Jaoa was less than0.5 cm, and mature individuals were up to 6 cm(Figs 2–4).

The thallus was composed of two or three layers. Thediameter of the outer cells was about 8–25 mm, smallerthan cells of the inner and middle layers (Fig. 5). Cellsof the middle layer were about 14–50 mm in diameter(Fig. 6), and the inner cells were 25–60 mm, biggerthan cells in the other two layers (Figs 7–9). The cellswere thin-walled with a single, laminate and parietalchloroplast that was discoid or band-shaped. The rhiz-oids attached to the substratum were formed byoutward elongation of outer layer cells at basal parts,which were colorless, with the cells being 3–6 mm inwidth, and 12–70 mm in length (Fig. 10).

Distribution: Jaoa was reported from temperate tosubtropical and tropical zones in China (Fig. 1). Typelocality: Beibei County, Chongqing province. (Jao 1941;Wang et al. 1988; Li & Hu 2003; Shi et al. 2003,2004).

Culture observation: Heterotrichous thalli were easilyobserved on solid medium, with infrequent and irregu-lar branches. After several weeks, prostrate filamentswith some short lateral branches became densely and

Figs 2–4. Jaoa prasina in the natural habitat, a stream below the

Nanchuan Reservoir, Xianning County, Hubei province, China.


3Phylogenetic position of Jaoa

centrally aggregated, displaying a pseudoparenchyma-tous aggregate structure. The inner cells of the aggre-gate were spherical or ellipsoidal, 20–40 mm indiameter, bigger in size than cells of external branches.The bigger cells protruded outward and then elongatedto form branched filaments, which were similar withrhizoids in the natural habitat (Figs 11–13). Therhizoid-like filaments radiated by elongation of severalshort and feeble branches. Most rhizoid-like cells werenarrow, 4–10 mm in width, 10–35 mm in length, with aparietal band-shaped chloroplast (Figs 11–13). Globu-lar aplanospores were formed with thickened walls andcould germinate into filamentous plants when condi-tions were favorable (Fig. 14). A vesicle-like thalluswithout any rhizoids developed when cultured in liquidmedium but was never observed on agar (Figs 15,16).

Phylogenetic analyses

Estimates of sequence similarity provided a simplemeasure of relationships between the green algal taxa.The SSU rDNA sequence from Jaoa prasina (Xianning)had 98.0%, 98.1%, 97.9%, 97.3%, and 97.5%sequence similarity with sequences of accessionnumbers. AY303594 and AY303595 (Acrochaeteviridis), JN104106 and AY303591 (Acrochaete lepto-chaete), FJ715684, AY303593 and AY303592(Acrochaete repens), JF680960 (Ulvella lens), andAY205328 (Acrochaete ramosa) obtained fromGenBank, respectively. The sequence similarity basedon partial rbcL DNA was 95.8% and 92.0% withsequences from Acrochaete repens (FJ715715) andUmbraulva amamiensis (AB097614), respectively.

Figs 5–10. Microscopy of Jaoa prasina

from the field. 5. Outer layer of

Jaoa prasina. 6. Middle layer of Jaoa

prasina. 7. Inner layer of Jaoa prasina

(Figs 5–7 are the same area under

different depth focus). 8. Longitudinal

section of Jaoa prasina showing the

thallus consisting of three layers.

9. Longitudinal section of Jaoa prasina

showing the thallus consisting of two

layers. 10. Rhizoid cells at the base of

the thallus. Bars = 50 mm.


4 H. Zhu et al.

Together with 23 previously published sequencesrepresenting the genera from the Ulvophyceae, the SSUrDNA sequences obtained in the present study wereused to reconstruct a phylogenetic tree (Fig. 17). ForrbcL sequences, 14 published sequences obtainedfrom GenBank were used to construct a phylogenetictree (Fig. 18).

The SSU rDNA phylogeny lacked support alongthe backbone but several terminal clades werewell-supported. The position of Jaoa prasina wasunresolved in the Ulvellaceae (Fig. 17). The rbcLphylogeny resolved Jaoa prasina as the sister taxon ofAcrochaete repens with 100% bootstrap values(Fig. 18).

DISCUSSION

Molecular phylogenetic analyses have dramaticallyreshaped our views of green algal relationships andevolution (Cocquyt et al. 2010). However, the phylo-genetic position of Jaoa Fan has never beenaddressed using sequence data. Initial molecular phy-logenetic investigations have generally corroboratedthe ultrastructure-based higher-level classification ofthe green algae, but have also revised the member-ship of several lineages (McCourt 1995), includingthe Ulvophyceae clade. For Jaoa Fan, Wang et al.(1988) found that the alga was very similar withUlvaceae in ultrastructural features.

Figs 11–16. Jaoa in culture. 11–

13. Filaments at an early stage on

agar. 14. Aplanospores germinating

on agar. 15. Filamentous aggregates

on agar. 16. The vesicle-like thallus in

liquid medium. Bars = 25 mm (Figs 13–

16); Bars = 5 mm in Figs 15–16.



Fig. 17. Phylogenetic tree constructed

from small subunit ribosomal DNA (SSU

rDNA) sequences of selected members

of the Ulvophyceae and Jaoa prasina.

Numbers at nodes represent bootstrap

support values for neighbor joining/

maximum parsimony/maximum likeli-

hood (NJ/MP/ML). Only values above 50

for NJ/MP/ML are shown. The sequences

obtained from our study are shaded gray.

Fig. 18. Phylogenetic tree constructed

from RuBisCO large subunit (rbcL) DNA

sequences of selected members of the

Ulvophyceae and Jaoa prasina. Numbers

at nodes represent bootstrap support

values for neighbor joining/maximum

parsimony/maximum likelihood (NJ/MP/

ML). Only values above 50 for NJ/MP/ML

are shown. The sequences obtained from

our study are shaded gray.


6 H. Zhu et al.

Species of the Ulvaceae are found in a wide range ofhabitats and environments, and they are notoriouslydifficult to identify because of the morphological plas-ticity expressed by many members, and the paucity ofreliable characters for differentiating taxa (Heeschet al. 2009). Species of Ulvella and Acrochaete com-monly attach epi- or endophytically on hard substrata,such as mollusc shells, stones, or red or green algae(Campo et al. 1998; Correa & Mclachlan 1991, 1992,1994; Deng et al. 2012). The morphological character-istics of Ulvella are described as follows: the plants arevery regularly disc-shaped and pseudoparenchymatous,they have a polystromatic central part and monostro-matic radiating filaments without rhizoids, and thewhole plant can be up to 2 mm (Smith et al. 1969;Nielsen 1977) with marginal cells frequently Y-shaped(Snow 1899). The morphological traits of the genusAcrochaete include a uniseriate thallus with branchedfilaments and central mutual compressed, cylindricalcells, and zoospores varying greatly in size betweendifferent species (Nielsen & Mclachlan 1986;Kornmann 1993; Deng et al. 2011). Gabrielson et al.(2006) transferred Endophyton ramosum Gardner, Epi-cladia flustrae Setchell & Gardner, E. codicola Setchell& Gardner, and Pseudopringsheimia apiculata Setchell& Gardner to Acrochaete. Cultural morphological fea-tures may provide some evidence of the taxonomyof Ulvellaceae. Many species were transferred tothe genus Acrochaete based on the production ofAcrochaete-type hairs in culture (Rinkel et al. 2012).

In our study the morphology of Jaoa in culture ismarkedly different from the field-collected thalli. Theinner inflated cells of plant mass observed in thepresent research were morphologically similar to innerlayer cells in plants in the natural habitat. The branchesformed by protuberance of inner inflated cells may behomologous with rhizoids that form in wild environ-ments. Additionally, the thallus cultured in liquidmedium can develop into vesicle-like plants, which isexactly similar to plants in natural habitats. In ourstudy, significant dissimilarity was evident in the grossmorphology of Ulvella, Acrochaete, and Jaoa in thenatural environment. However, the cultural morphologyof Jaoa in the present study was very similar with thecultural morphology of Acrochaete (Campo et al. 1998;Deng et al. 2012). Most zoospores of Ulvella and Acro-chaete are irregular ellipsoidal and flask-shaped inoutline according to Kornmann (1993) and Nielsen(1977). Moreover, spore germination of Acrochaetegeniculata is unipolar; the content of the spore migratesinto a well-defined germination tube, and after 1–2weeks, germlings appear as sparsely branched fila-ments (Campo et al. 1998). This resembles character-istics of Jaoa prasina observed by Jao (Jao 1941).

Jao thought the number of layers composing thethallus was the criterion for distinguishing species (Jao

1947). In our observation, the thallus of J. prasina wascomprised of two or three layers in the same individual.The internal transcribed spacer (ITS) sequences frommultiple geographically distinct collections of Jaoa(including J. prasina and J. bullata) were reported todiffer by only three to seven bases (Li & Hu 2003).Unfortunately, the ITS sequences were not published inany DNA database.

In the present study, Jaoa had the closest geneticrelationship with the genus Ulvella and Acrochaete inthe family Ulvellaceae based on SSU rDNA and rbcLsequences analyses (Figs 17,18). The topology of Ulvo-phyceae based on SSU rDNA sequences in our studywas generally congruent with that in O’Kelly et al.(O’Kelly et al. 2004), in which Jaoa clustered withAcrochaete repens, Acrochaete viridis, Acrochaete lep-tochaete, Acrochaete ramosa, and Ulvella lens. Bownet al. (2003) found that a paraphyletic group composedof only six species from the genus Acrochaete had theclosest relationship with Ulva and Enteromorpha.

Within the Ulvellaceae clade (Figs 17,18), there aresome smaller clades consisting of epilithic species suchas Jaoa, as well as epiphytic, endophytic, or endozoicspecies such as Acrochaete and Ulvella. Although Jaoa,Ulvella, and Acrochaete have substantial morphologicaldifferences, they had the closest phylogenetic relation-ship among Ulvophyceae in the present study. Jaoa,Ulvella, and Acrochaete together formed a cladestrongly supported in all analyses. The difficulty inidentifying morphological synapomorphies for clades inmolecular-based trees is not unique to the Ulvellaceaegroup. These results, combined with morphologicalobservations and culture studies, provide strong evi-dence that Jaoceae should not be recognized as aseparate family. Given that all genera in Ulvellaceaehad a close genetic relationship and passed through athread-like mass stage in development, except Ulvella,it is reasonable to propose changing the taxonomicposition of Jaoa from Jaoaceae to Ulvellaceae, and thefamily name Jaoaceae should be abandoned.

From the SSU phylogeny, Jaoa has a closer relation-ship with other species in Acrochaete than Acrochaeteendozoica, consistent with findings by Rinkel et al.(2012) and O’Kelly et al. (2004). However, we did notfind any Acrochaete-type hair on cultivated Jaoa speci-mens, which suggests Jaoa is a distinct genus fromAcrochaete and the systematic position of Acrochaeteendozoica should be revised.

Phylogenetic analysis indicated that Jaoa is in familyUlvellaceae, order Ulvales, which mostly containsspecies that live in ocean or saline water (only a fewspecies are freshwater). Ichihara studied Ulva lim-netica, a freshwater species, and held that Ulva lim-netica may have originated from marine ancestors, thenadapted into freshwater habitats and developed into anew species (Ichihara et al. 2009). As a genus close to



Ulva, Jaoa may also have split from a marine Ulvel-laceae population, and adapted to freshwater habitatsdue to geological movements or other reasons. Whileancestral populations in freshwater environmentsevolved into Jaoa, other populations remaining inmarine environments developed into Acrochaete orUlvella.

In the Ulvophyceae, morphologies range from micro-scopic unicells to macroscopic multicellular plants andgiant-celled organisms with unique cellular and physi-ological characteristics (Mine et al. 2008). There arefour main cytomorphological types (Cocquyt et al.2010; Leliaert et al. 2012). Based on the phylogeny of18S sequences, the genus Jaoa should be placed in thesecond type representing multicellular filaments orblades composed of uninucleate cells. The ultrastruc-tural features, which apply to most (but not all) greenalgae, were believed to accurately reflect phylogeneticrelationships because of their involvement in funda-mental processes of cell replication and cell motility,and thus lower likelihood for convergent evolution,compared to gross morphological traits (Leliaert et al.2012). For Jaoa Fan, a multicellular alga with a singlenucleus and chloroplast, Wang et al. (1988) found thatthe alga was very similar with Ulva mutabilis based onthe thylakoid arrangement and ultrastructural featuresof the pyrenoids. This result may provide importantinsights into the evolution of morphological complexityin the Ulvophyceae. The vesicle-like Jaoa and filamen-tous Acrochaete, with far greater morphological differ-ences, have the closest genetic relationship inferredfrom phylogenetic analyses. Thus, muticellularity mayhave evolved independently in the Ulvales-Ulotrichalesand TBCD (Trentepohliales, Bryopsodales, Cladopho-rales, Dasycladales) clades (Cocquyt et al. 2010).

ACKNOWLEDGMENTS

This work was supported the National Natural ScienceFoundation of China (No. 30870163, and 31270252).

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Phylogenetic position of Jaoa, a green algal genus endemic to China

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