Molecular Phylogenetics and Evolution 52 (2009) 329–339
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Phylogenetic relationships and species circumscription in Trentepohlia and Printzina (Trentepohliales, Chlorophyta)
Fabio Rindi *, Daryl W. Lam, Juan M. López-Bautista
Department of Biological Sciences, The University of Alabama, P.O. Box 870345, 425 Scientific Collections Bldg., Tuscaloosa, AL 35487-0345, USA article info a b s t r a c t
Article history: Subaerial green microalgae represent a polyphyletic complex of organisms, whose genetic diversity is Received 28 October 2008 much higher than their simple morphologies suggest. The order Trentepohliales is the only species-rich Revised 12 January 2009 group of subaerial algae belonging to the class Ulvophyceae and represents an ideal model taxon to inves- Accepted 15 January 2009 tigate evolutionary patterns of these organisms. We studied phylogenetic relationships in two common Available online 7 February 2009 genera of Trentepohliales (Trentepohlia and Printzina) by separate and combined analyses of the rbcL and 18S rRNA genes. Trentepohlia and Printzina were not resolved as monophyletic groups. Three main clades Keywords: were recovered in all analyses, but none corresponded to any trentepohlialean genus as defined based on 18S rRNA gene morphological grounds. The rbcL and 18S rRNA datasets provided congruent phylogenetic signals and Chlorophyta Molecular systematics similar topologies were recovered in single-gene analyses. Analyses performed on the combined 2-gene Morphological convergence dataset inferred generally higher nodal support. The results clarified several taxonomic problems and Printzina showed that the evolution of these algae has been characterized by considerable morphological conver- rbcL gene gence. Trentepohlia abietina and T. flava were shown to be separate species from T. aurea; Printzina lage- Subaerial algae nifera, T. arborum and T. umbrina were resolved as polyphyletic taxa, whose vegetative morphology Substitutional saturation appears to have evolved independently in separate lineages. Incongruence between phylogenetic rela- Trentepohlia tionships and traditional morphological classification was demonstrated, showing that the morphological Trentepohliales characters commonly used in the taxonomy of the Trentepohliales are phylogenetically irrelevant. Ulvophyceae Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction 2007). A direct consequence of this phenomenon is that the classi- fication at the genus and species level requires a reassessment Subaerial green microalgae are among the most widespread and based on a polyphasic combination of different approaches (Prösc- evolutionarily diverse organisms inhabiting terrestrial environ- hold and Leliaert, 2007). At present, the limited amount of DNA se- ments. Their systematics have been traditionally problematic and quence data available for most groups is the main hurdle to the surrounded by great controversy. In the past, their classification completion of such a reassessment. was entirely based on morphological characters (Smith, 1950; Ettl The order Trentepohliales is an ideal model taxon to illustrate and Gärtner, 1995). However, since the morphology of most spe- the complexity of the problems that affect the systematics and tax- cies is unicellular and affected by great plasticity, the set of reliable onomy of subaerial green algae. This order includes microchloro- characters useful for systematic purposes is limited. In the last phytes occurring in a wide range of habitats in areas with humid 15 years, DNA sequence data have become gradually available and rainy climates. Although most diverse and abundant in tropical and have led to important insights into the evolution of these regions, the Trentepohliales are also found in temperate zones and organisms. It has become clear that terrestrial green algae are a occur on a large variety of substrata, such as bark, leaves and fruits highly polyphyletic group originating from colonization of terres- of vascular plants, wood, metal, plastic, stones and concrete trial environments by many separate lineages of aquatic (both (Thompson and Wujek, 1997; López-Bautista et al., 2002; Rindi freshwater and marine) algae (Huss et al., 1999; Lewis and and López-Bautista, 2007). They are also among the most common McCourt, 2004; Lewis and Lewis, 2005; Watanabe et al., 2006; Le- phycobionts in lichens (Chapman and Good, 1983; Nakano and wis, 2007; Cardon et al., 2008). The adaptation to a terrestrial life- Ihda, 1996; Chapman and Waters, 2001). When present in large style has resulted into a convergent morphology in algae belonging amounts, their occurrence is revealed by the red, orange or yellow to widely separated evolutionary lineages (López-Bautista et al., color produced by the accumulation of carotenoids in their cells. The order contains the single family Trentepohliaceae and is distin- guished from all other green algae by the combination of the fol- * Corresponding author. Present address: Martin Ryan Institute, National Uni- versity of Ireland, Galway, Ireland. Fax: +353 91 525005. lowing features: presence of b-carotene and haematochrome; E-mail address: [email protected] (F. Rindi). absence of pyrenoids in the chloroplast; flagellar apparatus with
1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.01.009 330 F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339 unique ultrastructure (described in detail by Chapman and Henk, of variation between different character states in the same spe- 1983 and Roberts, 1984); transverse cell walls with plasmodes- cies, sometimes in the same specimen. Some species of Tren- mata; and presence of a characteristic reproductive structure, the tepohlia and Printzina are characterized by great plasticity sporangiate lateral (a highly modified branch consisting of an api- related to environmental factors, and their morphology may cal cell that is swollen basally and tapers apically into a short neck, vary considerably depending on the environmental conditions on which a spherical or oval zoosporangium is borne). (Howland, 1929; Nakano and Handa, 1984; Thompson and Wu- This unusual combination of characters has been a source of jek, 1997; Rindi and Guiry, 2002). As in other groups of ulvo- great confusion in the classification of the Trentepohliales at all phycean green algae (i.e., Verbruggen et al., 2007a; De Clerck taxonomic levels, in particular the class level. The possible inclu- et al., 2008), there are extensive ‘‘grey” zones between tradi- sion of these algae in at least three different classes was suggested tional species defined on morphology. Collection of specimens in the past (López-Bautista and Chapman, 2003, and references for which the morphology does not correspond satisfactorily therein), as well as the elevation to the rank of a class, the Tren- to any of the species currently included in these genera is a tepohliophyceae (Van den Hoek et al., 1995). López-Bautista and common situation in field investigations, and for this reason Chapman (2003) provided robust evidence for placement in the species of Trentepohlia are often identified at the genus level, class Ulvophyceae based on 18S rRNA sequence data. The molecu- sometimes only at family or order level. Another factor that lar evidence currently available suggests that the Trentepohliales greatly complicates taxonomic matters is the fact that several form a well-supported monophyletic group sister to a clade com- species were described by early authors, who provided only a posed of the order Dasycladales and the clade Cladophorales– very vague description (Linnaeus, 1753, 1759; Agardh, 1824; Siphonocladales (López-Bautista and Chapman, 2003). Kützing, 1843; Zeller, 1873); for some of them, no type speci- At the genus level, the classification of the Trentepohliales has mens or other authentic specimens are available (i.e., Trentepoh- been traditionally based on the scheme proposed by Printz lia aurea and T. iolithus). For this reason, the identity of some (1939). In this monograph, five genera (Cephaleuros, Phycopeltis, species has become very confused in the course of the time, Physolinum, Stomatochroon, Trentepohlia) were recognized on mor- and the circumscription of several morphologically similar taxa phological basis. Cephaleuros Kunze includes 17 species and con- remains to this day an unsolved matter. sists of filaments irregularly branched, free or coalescent to DNA sequence data for the Trentepohliales have become avail- produce irregular discs that grow below the cuticle or the epider- able relatively recently (López-Bautista and Chapman, 2003; mis of leaves of vascular plants. Species of Phycopeltis Millardet López-Bautista et al., 2003, 2006). López-Bautista et al. (2006) is consist of creeping filaments, usually coalescing to produce more thus far the only study that has considered the phylogeny of the or less regular discs epiphytic on leaves, stems and fruits of vascu- Trentepohliales at the genus and species level using molecular data lar plants; the genus currently includes 26 species. Stomatochroon (18S rRNA gene sequences). This investigation suggested a lack of Palm includes four species with diminutive habit, growing in correspondence between molecular phylogeny and traditional leaves of vascular plants as filamentous endophytes, the presence classification based on morphological characters, Cephaleuros of which is revealed by sporangiate laterals protruding out of the seemingly being the only monophyletic genus. These results sug- host plant’s stomata. Members of Trentepohlia Martius form small gested that subcuticular habit, heteromorphic life history and tufts or compact crusts consisting of numerous branched filaments occurrence of zoosporangia in clusters were the only morphologi- growing on a large variety of natural and artificial substrata; this is cal characters with potential phylogenetic significance. However, the largest and earliest-described genus, including about 40 spe- the use of a single-gene dataset along with a relatively limited tax- cies. Physolinum was erected by Printz (1921) for a single species, on sampling restricted the extent of the conclusions drawn in that Trentepohlia monilia De Wildeman, due to the production of apl- study. anospores; however, in the more recent literature the validity of Because of their morphological and ecological diversity and this character has been disputed and Physolinum has not been sep- widespread distribution, Trentepohlia and Printzina represent an arated from Trentepohlia (Flint, 1959; Cribb, 1970; Sarma, 1986). ideal subject to investigate the evolutionary mechanisms responsi- Printzina was erected by Thompson and Wujek (1992) for 9 taxa ble of morphological diversification in terrestrial eukaryotic micro- which were formerly included in Trentepohlia, and a newly de- algae. The objective of this study is to infer phylogenetic scribed species, P. ampla. Shape of zoosporangia, arrangement of relationships within Trentepohlia and Printzina through analyses the sporangiate laterals and development of the erect part of the of the nuclear-encoded 18S rRNA and the chloroplast-encoded thallus were the main characters on which Thompson and Wujek large subunit of the ribulose-1,5-bisphosphate carboxylase/oxy- (1992) based the separation of the two genera. genase (rbcL) genes. This investigation extends the dataset of 18S Taxonomy and systematics of Trentepohlia and Printzina have rRNA sequences available and provides the first published dataset been traditionally among the most complicated problems in the of rbcL sequences for these organisms. By comparing our molecular classification of the green algae. The evolutionary origins of these results with the morphology-based classification of the order Tren- organisms are obscure; it is presently unknown which morpholog- tepohliales, we provide new insights into the phylogeny of these ical characters bear phylogenetic significance and which character algae. states should be considered primitive and derived. In general, the taxonomy of these algae is still very unclear and in need of a de- 2. Materials and methods tailed reassessment. Morphological characters which have been used for taxonomic purposes include the following: shape and size 2.1. Collections, taxon sampling and vouchering of vegetative cells; relative development of the prostrate parts ver- sus the erect parts of the thallus; branching pattern; presence of Samples of Trentepohlia and Printzina were collected by the hair-like protrusions; arrangement of sporangiate laterals; shape authors in the course of previous studies or obtained from exter- and size of zoosporangium and enlarged cell bearing it (‘‘suffultory nal collaborators (Supplementary Table 1). Collections were cell”); arrangement of gametangia; shape of apical cells; features of made mainly in French Guiana (Rindi and López-Bautista, cell wall (such as color and presence of corrugations); and type of 2008), Panama (Rindi et al., 2008a), western Ireland (Rindi and substratum colonized (Hariot, 1890; De Wildeman, 1900; Printz, Guiry, 2002) and the Southeastern USA. The specimens were 1921, 1939; Sarma, 1986; Ettl and Gärtner, 1995). In practice, it stored in sealable plastic bags and identified at the best possible is not infrequent to observe a complete and uninterrupted range level of taxonomic resolution in the laboratory. Whenever possi- F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339 331 ble, the strains collected were isolated into unialgal cultures 2.4. Sequence alignment and phylogenetic analyses using liquid and agarized Jaworski’s Medium (Tompkins et al., 1995) and Bold’s Basal Medium with vitamins (Starr and Zeikus, Phylogenetic analyses were performed separately on the two 1987). Voucher specimens were deposited in the University of genes and on a combined 18S rRNA-rbcL dataset. For the 18S rRNA Alabama herbarium (UNA) and in the phycological herbarium gene, an alignment of 1618 basepairs was used for phylogenetic of the National University of Ireland, Galway (GALW). analyses. The alignment was constructed by adding sequences of Trentepohliales to the alignment of Siphonocladales of Leliaert 2.2. DNA extraction, PCR and sequencing et al. (2007), which was prepared on the basis of the secondary structure information, and adjusted by eye. The dataset approxi- DNA was extracted from 43 strains of Trentepohlia, Printzina mately corresponds to positions 78–1697 of the complete se- and Cephaleuros (Supplementary Table 1) using the Qiagen quence of Volvox carteri f. nagariensis X53904 (Rausch et al., DNeasy Plant Mini Kit (Qiagen, Valencia, California). With rela- 1989). Beside the newly sequenced strains, most of the 18S rRNA tively few exceptions (Trentepohlia cfr. abietina, T. dusenii, T. flav- sequences of Trentepohliales available in GenBank were used (Sup- a, T. umbrina from Tirrenia, Italy, and T. cfr. umbrina from French plementary Table 1). The choice of the outgroup taxa was based on Guiana) the extraction was performed on specimens isolated into recommendations by Verbruggen and Theriot (2008), and made unialgal cultures. For all PCR amplifications, a GeneAmp PCR after preliminary analyses in which different sets of taxa, more 2700 thermal cycler (Applied Biosystems, Foster City, California) and less closely related to the Trentepohliales, were used as out- was used. PCR of the 18S rRNA and rbcL genes was performed as groups. At present, the ulvophycean orders Cladophorales/ two overlapping fragments using several combinations of prim- Siphonocladales and Dasycladales are the groups which are known ers (Supplementary Table 2). Whereas most primers were ob- as most closely related to the Trentepohliales (López-Bautista and tained from the literature, some were designed by J.M.L.-B. Chapman, 2003). The preliminary analyses indicated that members using Oligo6.89 (Molecular Biology Insights, Cascade, Colorado). of these orders were a suitable choice as outgroup taxa, and the fol- Each 13 lL reaction contained: 4.4 lL of water; 1.25 lL of 10 lowing sequences were selected: Cladophora rupestris Z35319, Â reaction buffer; 1.25 lL of MgCl2 (25 mM); 1.25 lL of dATP, Cladophora vagabunda Z35316, Valonia utricularis Z35323, Bornetel- dCTP, dGTP and dTTP cocktail (8 mM); 0.625 lL of each primer la nitida Z33464 and Polyphysa peniculus Z33472. (10 mM); 0.1 lL of Taq (New England BioLabs, Ipswich, Massa- The rbcL alignment consisted of 879 basepairs (corresponding chusetts); 2.5 lL of betaine (5 M); and 1.0 lL of total genomic to the positions 232–1110 of the complete sequence of Chlorella DNA. For both 18S rRNA and rbcL, the normal PCR protocol con- vulgaris AB001684; Wakasugi et al., 1997) and was constructed sisted of an initial denaturing phase of 10 s at 96 °C, followed by by eye using MacClade 4.05 (Maddison and Maddison, 2002). No 40 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for indels were created in the rbcL alignment. The choice of the out- 1.5 min, with a final extension of 8 min at 72 °C. The PCR prod- group taxa was based on the same considerations as for 18S rRNA. ucts were examined for correct length, yield and purity under In this case, since no published rbcL sequences of Cladophorales/ UV light on 1% agarose gels stained with ethidium bromide, Siphonocladales are currently available, members of the Dasycla- and subsequently purified using the Qiagen MinElute Gel Extrac- dales and Bryopsidales were used as outgroup taxa: Batophora oer- tion Kit (Qiagen). The amount of DNA in PCR products was quan- stedii AY177748, Bornetella nitida AY177746, Polyphysa peniculus tified with a NanoDrop ND-1000 spectrophotometer (NanoDrop AY177743, Bryopsis hypnoides AY942169 and Halimeda discoidea Technologies, Wilmington, Delaware). PCR products were se- AB038488. The combined rbcL-18S rRNA dataset consisted of quenced in both directions using Big Dye version 3.1 (Applied 2496 basepairs and included 23 taxa for which both rbcL and 18S Biosystems) and sequence readings were obtained with an ABI rRNA could be sequenced successfully. Batophora oerstedii, Borne- 3100 automated sequencer (Applied Biosystems). Sequence chro- tella nitida and Polyphysa peniculus were used as outgroups. The matograms were aligned and contigs were created using Sequen- combined sequences used for these taxa were originally obtained cher 4.5 (Gene Codes Corporation, Ann Arbor, Michigan). from the same samples, sequenced for 18S rRNA by Olsen et al. (1994) and for rbcL by Zechman (2003). 2.3. Exploration of the characteristics of the molecular datasets Phylogenetic inference was based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). MP Three different methods were used in order to estimate the was performed using PAUP* 4.0b10; the analysis consisted of a relative amounts of phylogenetic signal and noise in the 18S heuristic search with random taxon addition (100 replicates), rRNA and rbcL datasets. First, skewness in the distribution of tree-bisection–reconnection branch swapping, steepest descent the data was examined by calculating the g1-statistic (Hillis and Multrees option enabled. For rbcL and the combined 18S and Huelsenbeck, 1992) using 10,000 random trees in PAUP* rRNA-rbcL dataset, the model-based analyses (ML and BI) were 4.0b10 ( Swofford, 1998). The values obtained were compared performed on partitioned datasets, applying separate models to with the empirical threshold values reported by Hillis and Huel- each partition. Three partitions were used for rbcL (first, second senbeck (1992) to verify the non-random distribution of the and third codon position); four partitions were used for the com- data. Second, potential substitutional saturation in the datasets bined dataset (the three codon positions of rbcL and the whole analyzed was evaluated graphically by plotting the uncorrected 18S rRNA gene). Rate differences between partitions were accom- p-distances against the Maximum Likelihood-corrected distances modated with ‘‘rate amplifier” parameters in the models of mod- as determined with the evolutionary model which yielded the el-based analyses, using the default settings of the programs best fit to the data (estimated using the corrected Akaike Infor- used. ML was performed using Treefinder (version April 2008; Jobb mation Criterion (AICc) in ModelTest 3.7; Posada and Crandall, et al., 2004). The models and parameters selected by Treefinder 1998). Separate plots were produced for datasets with and with- were applied. For 18S rRNA, the model selected by Treefinder un- out outgroup taxa. Finally, a test of substitutional saturation der the AICc criterion was GTR + G; for rbcL, the models selected based on the ISS statistic, as proposed by Xia et al. (2003) and were GTR + G for the first codon position, TVM + G for the second implemented in DAMBE (Xia and Xie, 2001), was performed sep- position and J1 + G for the third position. For MP and ML, nodal arately for the 18S rRNA and rbcL datasets, as well as for the support was assessed by non-parametric bootstrap analysis (Fel- three codon positions of rbcL separately. senstein, 1985) with 1000 resamplings. 332 F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339
BI was performed using MrBayes 3.04 (Huelsenbeck and Ron- uncorrected p-distances, suggesting little saturation (Fig. 1A and quist, 2001). In each BI analysis, two parallel runs of four Monte B). In the rbcL gene, similarly limited saturation was observed for Carlo Markov Chains (one cold and three incrementally heated) the first and second codon positions, as well as for the whole gene were conducted for 2,000,000 generations, with tree sampling (Fig. 1C–H). As expected, evidence of partial saturation was ob- every 100 generations. Priors and probability proposals set as de- served for the third codon position, for which the plot tended to le- fault in MrBayes were used. The stationary distribution of the runs vel off with increasing genetic distance (Fig. 1I and J). However, the was verified before to stop the analysis; it was assumed that the majority of the parsimony-informative positions in rbcL were lo- stationary distribution was reached when the average standard cated in the third position (226 out of 327, =69%) and analyses in deviations of split frequencies between the two runs was lower which the third position was excluded did not improve phyloge- than 0.01. If the stationary distribution was not reached at the netic resolution. MP and ML analyses performed only on first and end of the runs, the analysis was continued for additional second positions resulted in mostly unresolved trees; although 1,000,000 generations. The burn-in phase was assessed by plotting the number of most parsimonious trees recovered was relatively the number of generations against the likelihood scores and deter- low (seven with outgroups, three without outgroups), the boostrap mining where the analysis reached stationarity; 100,000 to analyses recovered very few well-supported nodes. Similar results 300,000 generations were discarded as burn-in, and the remaining were obtained in analyses performed on the translated amino acid samples were used to construct the majority-rule consensus trees. sequence, in which large polytomies and few resolved nodes oc- For each partition, the model was set to GTR + I + G by setting the curred (results not shown). Analyses performed only on the third number of substitution types to 6 with gamma shape parameter position recovered three most parsimonious trees with outgroups and proportion of invariable sites; the parameters were unlinked and five without outgroups. Although they were not able to resolve and allowed to vary across partitions. the internal nodes, they provided a higher number of supported In relevant cases, the significance of differences between the nodes and their results were generally more similar to analyses topologies inferred in the analyses and other possible topologies performed on the whole gene. The results of the saturation tests was tested using Shimodaira–Hasegawa (SH) tests (Shimodaira based on the ISS statistic did not indicate substantial saturation. and Hasegawa, 1999) or Approximately Unbiased (AU) tests (Shi- The ISS.C values were significantly lower than the ISS values, when modaira, 2002) as implemented in Treefinder. calculated by DAMBE for both symmetrical and asymmetrical trees (indicating little saturation; Table 1). Only the test performed on 3. Results the third position of rbcL in the dataset inclusive of outgroups gave not significant results for a highly asymmetrical tree (suggesting 3.1. Characteristics of the molecular datasets substantial saturation in the case of a highly asymmetrical tree); however, the results of the same test were significant in the case In the Trentepohliales, the rbcL gene showed a considerably of a symmetrical tree. higher substitution rate than the 18S rRNA gene (Table 1). In the ingroup taxa, 387 variable sites (44%) occurred in the rbcL align- 3.2. Phylogenetic analyses ment; 327 of these (37%) were parsimony-informative. Only 175 variable sites (=10.8%) were found in the 18S rRNA alignment, The topologies recovered in the rbcL and 18S rRNA trees were 117 of which (7.2%) were parsimony-informative. The highest p- generally congruent. No major discrepancies occurred between dif- uncorrected sequence distances were 21.1% for rbcL (between ferent methods of phylogenetic reconstruction; differences were Printzina lagenifera from French Guiana and Trentepohlia umbrina only observed in the placement of taxa or clades that received little from Pavia, Italy) and 5.5% for 18S rRNA (between Trentepohlia or no statistical support. umbrina from Ireland and Physolinum monile from Louisiana State In general, rbcL showed better resolution than 18S rRNA, recov- University culture collection). For all datasets, with and without ering a higher number of well-supported nodes (BP > 60%, outgroup taxa, the g1-statistic was considerably more negative PP > 0.9). In the rbcL phylogeny, three main clades were recovered (left-skewed) than the empirical threshold values of Hillis and with moderate to high support (Fig. 2). A clade sister to all other Huelsenbeck (1992), indicating that the alignments were signifi- Trentepohliales (hereafter indicated as clade 1; Fig. 2) included cantly more structured than random data (suggesting limited sat- species of Trentepohlia and Printzina with very different morphol- uration). In 18S rRNA, the saturation plot for the ingroup taxa ogy and widespread geographical distribution (such as Printzina showed a near-linear correlation between ML-corrected and bosseae, Trentepohlia flava, T. iolithus and T. umbrina), as well as
Table 1
Characteristics of the datasets analyzed. Figures without brackets refer to datasets without outgroups; figures in brackets refer to datasets including outgroups. For the ISS statistic, tests marked with an asterisk indicate significant results (=indicative of little saturation); the ISS.C values indicated are the values calculated by DAMBE assuming symmetrical topologies.
rbcL (total) rbcL (1st position) rbcL (2nd position) rbcL (3rd 18S rRNA rbcL + 18S rRNA position) Characters analyzed 879 293 293 293 1618 2496 Parsimony-informative sites 327 (374) 70 (87) 31 (40) 226 (247) 117 (424) 380 (644) Variable parsimony-uninformative sites 60 (56) 17 (21) 11 (15) 32 (20) 58 (142) 95 (188) Invariable sites 492 (449) 206 (185) 251 (238) 35 (26) 1443 (1052) 2021 (1664) Highest uncorrected p-divergence within 21.1% 5.5% 10.0% ingroup taxa Number of most parsimonious trees/length in 3/1584 4/398 2/1257 steps (5/2061) (31/1049) (1/2009) Measure of skewness 0.559123 0.461276 0.397632 0.571262 0.751748 0.973715 À À À À À À (g -value) ( 0.480546) ( 0.442506) ( 0.382366) ( 0.478957) ( 1.985496) ( 1.394987) 1 À À À À À À ISS statistic 0.327/0.726* 0.204/0.659* 0.152/0.659* 0.431/0.659* 0.309/0.779*
(ISS/ISS.C, value for 32 taxon data subsets) (0.325/0.695)* (0.222/0.676)* (0.147/0.659)* (0.480/0.675)* (0.194/0.778)* F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339 333
0.3 0.06
0.25 A 0.05 B
0.2 0.04
0.15 0.03
0.1 0.02 Uncorrected p-distances Uncorrected Uncorrected p-distances Uncorrected 0.05 1 S8 htiw( spuorgtuo ) 0.01 81 S (without outgroups) 0 0 0 0.2 0.4 0.6 0.8 1 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 ML-corrected distances ML-corrected distances
0.3 0.25
0.25 C D 0.2 0.2 0.15 0.15 0.1 0.1
Uncorrected p-distances Uncorrected 0.05 0.05 p-distances Uncorrected rbcL (with outgroups) rbcL (without outgroups) 0 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 0.1 0.2 0.3 0.4 0.5 ML-corrected distances ML-corrected distances
0.25 0.18 E 0.16 0.2 F 0.14 0.12 0.15 0.1 0.08 0.1 0.06
0.05 0.04 Uncorrected p-distances Uncorrected st p-distances Uncorrected st rbcL 1 (with outgroups) 0.02 rbcL 1 (without outgroups) 0 0 0 0.1 0.2 0.3 0.4 0.5 0 0.05 0.1 0.15 0.2 0.25 0.3 ML-corrected distances ML-corrected distances
0.12 0.09 0.08 0.1 G H 0.07 0.08 0.06 0.05 0.06 0.04 0.04 0.03 0.02 Uncorrected p-distances Uncorrected 0.02 p-distances Uncorrected rbcL 2nd (with outgroups) 0.01 rbcL 2nd (without outgroups) 0 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 ML-corrected distances ML-corrected distances
0.6 0.5 I 0.45 J 0.5 0.4 0.35 0.4 0.3 0.3 0.25 0.2 0.2 0.15 0.1 Uncorrected p-distances Uncorrected Uncorrected p-distances Uncorrected 0.1 rbcL 3rd (with outgroups) rbcL 3rd (without outgroups) 0.05 0 0 0 0.5 1 1.5 2 2.5 3 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 ML-corrected distances ML-corrected distances
Fig. 1. Analysis of saturation of the 18S rRNA and rbcL genes by plotting ML-corrected distances against uncorrected p-distances. Graphs in the left column are for databases with outgroups; graphs in the right column are for databases without outgroups. The databases concerned are specified for each graph. 334 F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339
paraphyletic by a strain of Trentepohlia dialepta from CCAP culture collection (the identity of which, however, is unclear; see Section 4). Some additional taxa, such as Trentepohlia aurea (type species of Trentepohlia), Trentepohlia cfr. abietina from Spain and Printzina sp. (F223) did not belong to any of the three main clades; due to the lack of support in the internal branches, their position relative to the other clades could not be clarified. Despite lower resolution, the 18S rRNA dataset provided phylo- genetic patterns in agreement with the rbcL results (Fig. 3). The rel- ative positions of individual taxa were nearly identical and no major differences between the two phylogenies were found. The same three main clades were present; only the clade 3 (the Ceph- aleuros-clade), however, received strong nodal support. The clades 2 and 3 and Trentepohlia aurea were clustered in a common clade with high statistical support; the position of Trentepohlia aurea and Printzina sp. (F233) was again unresolved. Some additional species for which rbcL could not be sequenced were included in the 18S rRNA phylogeny. Trentepohlia abietina was placed in the clade 1, sister to a clade formed by three strains of Trentepohlia umbrina; Physolinum monile occurred in the clade 2, where it was recovered with high support as sister taxon to a strain of Printzina cfr. lagenifera from Hawaii. Analyses carried out on the combined dataset 18S rRNA-rbcL in- cluded 23 taxa and provided the results with the highest BP and PP
Fig. 2. Phylogram inferred from Maximum Likelihood analysis of the rbcL gene in Trentepohlia, Printzina and related taxa, with bootstrap support (BP) and Bayesian posterior probabilities (PP) indicated at the nodes. From left to right (or from top to bottom, above and below branches), support values at nodes correspond to Maximum Parsimony BP, Maximum Likelihood BP and Bayesian PP. BP values lower than 60% and PP lower than 0.85 are not reported. The tree was rooted using outgroup sequences of Dasycladales and Bryopsidales (not shown) specified in Section 2. some tropical strains of Trentepohlia that could not be identified with certainty at the species level. The relative positions of taxa in this clade were consistent between different analyses, but not always supported by high BP values and Bayesian PP; a well-sup- ported separation, however, was found between some strains of Trentepohlia (T. flava, T. iolithus, T. umbrina and T. cfr. umbrina) and the other taxa within the clade (Fig. 2). The other Trentepohli- ales sequenced belonged mainly to two relatively well-supported clades. One of these (hereafter indicated as clade 2; Fig. 2) included numerous strains of Trentepohlia and Printzina, collected mostly in the tropics. The vegetative morphology of these algae corre- sponded primarily to two widely distributed tropical species, Printzina lagenifera (type species of Printzina) and Trentepohlia arborum. In the case of Printzina lagenifera, some strains could not be identified with certainty at the species level because they did not become reproductive; it was therefore impossible to ob- serve the flask-shaped gametangia, which represent the diagnostic character of this species. Neither Printzina lagenifera nor Trentepoh- Fig. 3. Phylogram inferred from Maximum Likelihood analysis of the 18S rRNA lia arborum produced well-supported monophyletic groups and in gene in Trentepohlia, Printzina and related taxa, with bootstrap support (BP) and general reconstruction of relationships in this clade was impossi- Bayesian posterior probabilities (PP) indicated at the nodes. From left to right ble, since almost none of the basal nodes received any support. support values at nodes correspond to Maximum Parsimony BP, Maximum The other clade (hereafter indicated as clade 3; Fig. 2) was roughly Likelihood BP and Bayesian PP. BP values lower than 60% and PP lower than 0.85 are not reported. The tree was rooted using outgroup sequences of Cladophorales correspondent to the genus Cephaleuros. It included several strains and Dasycladales (not shown) specified in Section 2. New sequences produced in of this genus obtained from culture collections, but it was rendered the present study are marked with an asterisk. F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339 335 support (Fig. 4). In general, all clades which received support in tains a good deal of phylogenetic signal (Gontcharov et al., 2004; single-gene analyses were recovered with higher support in the De Clerck et al., 2006). Usually, analyses performed on the first combined analyses. The three main clades were again recovered. and second positions provide a high number of most parsimonious As inferred in the 18S rRNA phylogeny, the clades 2 and 3 and Tren- trees and poor resolution, whereas analyses limited to third posi- tepohlia aurea and Printzina sp. (F223) occurred in a common, tions yield results similar to analyses performed on the whole gene highly-supported clade. However, relationships within the clade (Lewis et al., 1997; McCourt et al., 2000; Zechman, 2003; Gontcha- 2 were again problematic, as most internal nodes were unresolved; rov et al., 2004; De Clerck et al., 2006). The rbcL dataset analyzed in once again, Printzina lagenifera was not monophyletic. The position this study shows a higher substitution rate than in other groups of of Trentepohlia aurea was well resolved in the Bayesian analysis, in green algae (a likely reflection of this high variability is the fact which this species was sister to Cephaleuros with high PP; such that it has proved impossible to design rbcL primers working satis- relationship, however, received no support in the MP and ML boot- factorily for all trentepohlialean taxa on the whole length of the strap analyses. The position of Printzina sp. (F223) was again gene). Parsimony-informative positions account for 37% of the to- unresolved. tal sites, whereas in most green algal orders they usually range be- tween 25% and 30% (Zechman, 2003; Lam and Zechman, 2006; 4. Discussion Rindi et al., 2007, 2008b). Third positions include 69% of the parsi- mony-informative sites in our dataset and the distance plots show 4.1. Characteristics of the molecular datasets evidence of saturation, especially when the outgroups are in- cluded. At the same time, however, analyses performed only on Previous trentepohlialean molecular datasets were limited to a third positions yielded a higher number of resolved nodes than relatively low number of 18S rRNA and phragmoplastin gene se- analyses performed on the first and second positions. The g1 scores quences (López-Bautista and Chapman, 2003; López-Bautista and the ISS values calculated for the whole gene suggest that the ef- et al., 2003, 2006). The results presented here are in agreement fect of the third position does not cause substantial harm to the with previous studies based only on 18S rRNA sequences and the phylogenetic informativeness of the dataset. In consideration of topology of our trees corresponds well with that of López-Bautista this, and of the fact that the phylogenetic signals provided by rbcL et al. (2006), which was based on a more limited taxon sampling. and 18S rRNA are largely congruent, we regard the retention of Compared to these data, rbcL sequences were expected to provide rbcL third positions as an appropriate solution for the aims of the better resolution at the genus and species level, and the results of present investigation. The combination of these two genes has this study have basically confirmed this prediction. Among algae, proved to be a powerful tool of inference at several taxonomic lev- the rbcL gene has proved very useful for testing phylogenetic els, extracting successfully the phylogenetic signal of the fast- hypotheses at a range of different taxonomic levels, even when evolving rbcL and resolving better shallow evolutionary relation- only partial sequences are used. As a protein-coding gene, it is ships, where 18S rRNA lacks variability (Gontcharov et al., 2004). potentially affected by problems of substitutional saturation at In our case, although not resolutive for several internal nodes the third codon (wobble) position. In some studies this has been (especially in the clade 2), the use of the combined dataset repre- regarded as a major impediment to phylogenetic inference, and sents an improvement and provides to most nodes higher support the third positions have been eliminated from the analyzed data- than single-gene analyses. This situation was observed in almost sets (Nozaki et al., 2000; Nakada et al., 2008). However, the major- every study in which rbcL and 18S rRNA were combined (e.g., Hep- ity of the rbcL-based investigations has concluded that, even when perle et al., 1998; Hayden and Waaland, 2002; Gontcharov et al., the third position shows evidence of partial saturation, it still con- 2004; Nakada et al., 2008).
4.2. Implications for the systematics of Trentepohlia and Printzina
Our molecular analyses revealed a phylogenetic scenario in sub- stantial contrast with the traditional taxonomic scheme of Tren- tepohlia and Printzina based on morphology (Printz, 1939). Members of Trentepohlia and Printzina were present in all three major clades recovered in our phylogenies. None of these clades corresponded with the current morphological circumscription of any trentepohliacean genus, with the only partial exception of Cephaleuros. All strains of Cephaleuros sequenced were clustered in a clade that received high support in all three datasets analyzed and under all methods of inference used. Cephaleuros was mono- phyletic in the 18S rRNA and combined analyses, but in the rbcL phylogeny a strain of Trentepohlia dialepta from CCAP made it para- phyletic. This suggests that T. dialepta should be transferred to Cephaleuros. Trentepohlia dialepta was originally described by Nylander (1862) as Coenogonium dialeptum using material from Brazil. As Cephaleuros, this species is widespread on leaves of trees in tropical regions (Hariot, 1889a; Printz, 1939) and shares with this genus some important morphological similarities, in particular the habit of the erect filaments and the arrangement of the zoospo- rangia in clusters at the top of the erect axes (Salleh and Milow, Fig. 4. Phylogeny of Trentepohlia, Printzina and related taxa inferred from Maximum 1999). It is noteworthy, however, that the strain from CCAP was Likelihood analysis of the combined dataset rbcL-18S rRNA. From left to right isolated from material from Kampala, Uganda, ‘‘thought to be a cof- support values at nodes correspond to Maximum Parsimony BP, Maximum fee parasite” (http://www.ccap.ac.uk/strain_info.php?Strain_- Likelihood BP and Bayesian PP. BP values lower than 60% and PP lower than 0.85 are not reported. The tree was rooted using outgroup sequences of Dasycladales No=483/2). Species of Cephaleuros grow in tropical and (not shown) specified in Section 2. subtropical regions as subcuticular epiphytes on leaves of many 336 F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339 plants, including coffee (Chapman, 1984; Thompson and Wujek, and so far we have been unable to obtain reliable sequences from 1997; Chapman and Waters, 2001). Therefore, the possibility that this genus. Additionally, the type species Phycopeltis epiphyton the strain in question is a Cephaleuros which was misidentified in (Millardet, 1870) is one of the smallest-sized in the genus and first instance cannot be excluded. It is also worth mentioning that can be detected with the unaided eye only when it produces large species of both Trentepohlia and Cephaleuros, when growing in cul- populations. The same considerations apply to Stomatochroon, ture, assume a free filamentous morphology and become almost which, for its small size and elusive nature (this alga lives inside indistinguishable. of the substomatal chambers of plant leaves), is the least recorded The large majority of the strains of Trentepohlia and Printzina se- trentepohlialean genus (Thompson and Wujek, 1997; López-Bau- quenced belonged to either clade 1 or clade 2. Both clades include tista et al., 2002). In the course of our field surveys, we have never strains with very different morphology, and it is presently impos- found specimens of this genus. sible to determine any morphological synapomorphies defining these two clades. It is clear that none of the morphological charac- 4.3. Morphological versus phylogenetic species delineation in ters traditionally used for taxonomic purposes (Hariot, 1890; De Trentepohlia and Printzina Wildeman, 1891, 1900; Printz, 1939; Sarma, 1986; Ettl and Gärt- ner, 1995) are phylogenetically significant. Ecological traits such Our molecular results indicate that the circumscription of as type of habitat and substratum have also been used to distin- species in Trentepohlia and Printzina is a difficult matter. The corre- guish species and groups of species in the Trentepohliales (Printz, spondence between morphological and phylogenetic circumscrip- 1939). In particular, in the original description of Printzina, Thomp- tion varies greatly from species to species and needs to be son and Wujek (1992) reported the type of habitat as one of the evaluated case by case; it is clear that a species concept based only main characters distinguishing this genus from Trentepohlia on morphological features is inadequate for the Trentepohliales (as (Printzina associated with shaded forest habitats, Trentepohlia for most other groups of terrestrial green algae) and the use of occurring at more exposed sites). Our results show that these char- molecular data will play an essential role in the taxonomy of these acters are also phylogenetically irrelevant, since they are not asso- algae in the future. Given the morphological homoplasy revealed ciated with any well-supported monophyletic group; algae by our results, the availability of a molecular marker suitable for growing on both natural and artificial substrata and in both ex- DNA barcoding would be an ideal solution for species definition posed and sheltered habitats are mixed together in both clades. in the Trentepohliales. We feel that the substitution rate of rbcL Geographical distribution may be in part a better indicator of phy- would be adequate for DNA barcoding in this order, and in fact logenetic relationships. Whereas most of the strains of the clade 2 other studies have suggested the suitability of rbcL for this purpose were identified as Printzina lagenifera or Trentepohlia arborum, two in other groups of Ulvophyceae (Verbruggen et al., 2007b). How- widespread tropical and subtropical algae (De Wildeman, 1891, ever, easy amplification through primers designed from conserved 1900; Printz, 1939; Rindi et al., 2005, 2008a), species associated sites is a fundamental requirement for barcoding purposes, and in mainly with temperate regions (Trentepohlia abietina, T. flava, T. io- the case of the Trentepohliales rbcL does not satisfy such require- lithus and T. umbrina) belong to the clade 1. However, exceptions to ment. The preparation of our database required the use of multiple this situation are present in both clades (e.g., Printzina bosseae in couples of primers, and several samples could not be amplified at the clade 1, Printzina cfr. lagenifera from Italy and P. lagenifera from all. In general, the development of molecular markers for DNA bar- Ireland in the clade 2) and we feel that further taxon sampling is coding is still a highly debated topic for green algae and plants; to necessary before drawing generalizations in this regard. Some date there is not yet a marker which has been accepted as a univer- additional taxa (Trentepohlia aurea, T. cfr. abietina from Spain and sal DNA barcode (Kress and Erickson, 2008) and intensive work in Printzina sp. F223) are not associated with any of the three main this area is still going on. clades and their positions were not fully resolved in any of the For some species with relatively stable and well-defined datasets analyzed. In the case of Trentepohlia aurea, this is particu- morphology, all strains included in our analyses produced well- larly problematic, since this is the type species of Trentepohlia. supported clades. Conversely other taxa, as delineated on morpho- Therefore, its position affects directly the genus-level classification logical basis, appear to be paraphyletic or polyphyletic entities that in the whole order. are better regarded as complexes of cryptic species. Trentepohlia At present, the genus-level taxonomy in the Trentepohliales is aurea represents a good example of the first case. This species, based entirely on morphology. The subdivision of the six genera which is the generitype of Trentepohlia, has always been relatively is straightforward and founded on easily observable characters, well defined from a morphological point of view (e.g., Martius, such as habit, morphology of the erect parts relative to the pros- 1817; Kützing, 1854; Hansgirg, 1886; Hariot, 1889b; Printz, trate parts, and arrangement of the sporangiate laterals (Printz, 1939) and the morphology of the numerous specimens deposited 1939). Thompson and Wujek (1992, 1997) introduced some addi- in European herbaria under this name is generally constant (Fabio tional characters related to the morphology of the zoosporangia Rindi, personal observation). Our molecular data confirm its sepa- (shape of the zoosporangium and position of the escape pore). ration from similar species, such as Trentepohlia abietina and T. flav- Our molecular analyses present strong evidence that such classifi- a, which has been very controversial in the past (e.g., Hariot, cation will need major modifications based on phylogenetic princi- 1889b; De Wildeman, 1900; Printz, 1921, 1939; Cribb, 1970). ples. Designations at the genus level might be an appropriate Conversely, Trentepohlia umbrina, T. arborum and P. lagenifera solution for our clades 1, 2 and 3. This, however, would introduce represent polyphyletic morphotaxa forming complexes of cryptic radical rearrangements in the nomenclature and in the genus cir- species evolved through morphological convergence. For Tren- cumscription of the Trentepohliales; we feel that in the present sit- tepohlia umbrina, the European samples that we sequenced form uation this would be premature. Decisions on these matters should a well-supported monophyletic group (Fig. 3) and we consider be made only after additional molecular datasets able to resolve them genuinely representative of this species (described by Küt- the relationships of lineages eligible as genera (in particular the po- zing (1843) for corticolous material from southern Germany). sition of Trentepohlia aurea relative to the other main clades) be- However, an identical or very similar morphology was also found come available. Even more important will be the availability of in a sample from Louisiana belonging to the clade 2 and a sample high-quality sequences of two genera that we could not include from French Guiana which, although occurring in the clade 1, was in our databases, Phycopeltis and Stomatochroon. Unfortunately, separated from European T. umbrina with high support (Fig. 2). For species of Phycopeltis are virtually impossible to grow in culture Trentepohlia arborum and Printzina lagenifera the situation is more F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339 337 complicated. Specimens morphologically corresponding to these and has delayed a full appreciation of their genetic diversity. While species occur in the clade 2, which in the overall scenario of tren- cases of morphological convergence have already been docu- tepohlialean evolution represents a very problematic lineage. In mented for species of the classes Trebouxiophyceae and Chloro- this clade most internal branches are very short when compared phyceae (Huss et al., 1999; Rindi et al., 2007), this study and our to the terminal branches, and the support for most internal nodes recent discovery of the genus Spongiochrysis (Rindi et al., 2006) is non-existent in all datasets analyzed. Reducing the length of ter- show that this phenomenon has also taken place for terrestrial minal branches by adding more taxa may be expected to benefit members of the Ulvophyceae. The overall message emerging from future phylogenetic reconstructions. But the fact that the same these studies is that life in terrestrial environments involves some patterns are observed in two genes with very different characteris- constraints for which a diminutive size and the reduction to a lim- tics (such as rbcL and 18S rRNA) suggests that the short internal ited range of morphological forms are favored. At present, it is branches are the result of a fast evolutionary radiation, and ulti- impossible to establish which environmental factors are the main mately only the use of additional molecular datasets (such as ITS constraints forcing such reduction and convergence (and the and the D1–D2 28S rRNA) may provide better resolution. For these almost complete absence of fossil record represent a substantial reasons, we cannot exclude that the inclusion of further loci in our limitation to propose hypotheses in this regard). However, we feel molecular phylogeny would eventually resolve Printzina lagenifera that a combination of systematic studies with physiological, bio- and Trentepohlia arborum as monophyletic taxa. However, for rbcL chemical and ecological investigations (focusing in particular on an average uncorrected p-sequence distance of about 10% was ob- dispersal mechanisms) might lead to major insights into this inter- served between strains morphologically corresponding to Printzina esting aspect of terrestrial algal evolution. lagenifera (with a maximum of 11.38% between P. lagenifera from Panama and P. lagenifera from Ireland). A similar situation was ob- Acknowledgments served between strains morphologically corresponding to Tren- tepohlia arborum, for which an uncorrected p-sequence distance The study was funded by the US National Science Foundation as high as 10.43% (between T. arborum from French Guiana and (Systematics Program DEB-0542924 to J.M.L.-B.), with partial T. arborum from Panama) was found. Furthermore, we performed support from the College of Arts and Sciences and the Depart- SH and AU tests in order to test differences between the trees ment of Biological Sciences, The University of Alabama. F.R. is recovered by our ML analyses and trees in which the strains mor- grateful to the Marine Institute of Ireland for financial support phologically referable to Printzina lagenifera and Trentepohlia arbo- under the Beaufort Biodiscovery Programme. D.W.L. acknowl- rum were constrained into monophyletic groups. For all three edges the University of Alabama for financial support in the form databases (rbcL, 18S rRNA, rbcL + 18S rRNA) the tests showed sig- of Aquatic Biology and Graduate Council Fellowships. Assistance nificant differences, rejecting the monophyly of P. lagenifera and from Britton O’Shields with DNA extractions and PCR, as well T. arborum. Such evidence suggests that P. lagenifera and T. arborum as from Brook Fluker and Wally Holznagel with DNA sequencing, are unlikely to be individual species, and more probably represent were greatly appreciated. We are very grateful to Frederik Lelia- complexes of cryptic species with convergent morphology. This ert for stimulating discussion, suggestions which greatly helped will make necessary a precise characterization of their taxonomic to improve the manuscript, and the 18S rRNA alignment of identities, which in the case of Printzina lagenifera will be particu- Siphonocladales that was used for the preparation of the 18S larly difficult. This species was described by Hildebrand (1861) rRNA alignment. Recommendations by Heroen Verbruggen and from bark of vines in a greenhouse hosting palms in Germany. Al- an anonymous reviewer were of great help to improve the origi- most certainly, the material used by Hildebrand for the description nal version of the manuscript. Assistance by Frédéric Lecorre was introduced as epiphyte on tropical plants; without knowledge (Floramazone) during fieldwork in French Guiana and by the staff of the vector plant’s origin, it is impossible to know the actual ori- of the S.T.R.I. Station of Barro Colorado Island (in particular Oris gin of the alga. Acevedo) during fieldwork in Panama is gratefully acknowledged. Collections of Trentepohliales received from Ricardo Tsukamoto, 4.4. Conclusions Alison Sherwood, Michael Guiry, Benito Gómez-Silva, Patrick Martone, Willem Prud’homme Van Reine and Paul Broady were Terrestrial algae of the genera Trentepohlia and Printzina repre- greatly appreciated. Dr. Bruno De Reviers is gratefully acknowl- sent a group of organisms in which morphological traits are not edged for the loan of an authentic specimen of Trentepohlia duse- indicative of evolutionary relationships. These genera do not form nii (PC0110846) from PC. monophyletic groups and their separation is not justified by molecular data. If their separation is to be maintained, their mor- phological circumscription will have to be reassessed radically. Appendix A. Supplementary data The morphology of these algae is affected by great homoplasy, which has caused convergence of separate taxa into an almost Supplementary data associated with this article can be found, in identical morphology. Because of this, the concept of species in the online version, at doi:10.1016/j.ympev.2009.01.009. Trentepohlia and Printzina (and in the Trentepohliales in general) cannot be based exclusively on morphological grounds. The taxo- nomic identity of each species will have to be circumscribed case References by case, using a combination of molecular data and morphological Agardh, C.A., 1824. Systema Algarum. Berling, Lund. investigations in the field and in culture. We also feel that other Cardon, Z.G., Gray, D.W., Lewis, L.A., 2008. The green algal underground: types of genotypic and phenotypic characters (chromosome num- evolutionary secrets of desert cells. Bioscience 58, 114–122. ber, secondary metabolites, ultrastructural characteristics) deserve Chapman, R.L., 1984. An assessment of the current state of our knowledge of the Trentepohliaceae. In: Irvine, D.E.G., John, D.M. (Eds.), Systematics of the Green to be investigated in depth, as they could reveal the synapomor- Algae. Academic Press, London, pp. 233–250. phies which cannot be found in the morphology. Chapman, R.L., Good, B.H., 1983. Subaerial symbiotic green algae: interactions with Finally, it is becoming increasingly obvious that the morpholog- vascular plant hosts. In: Goff, L.J. (Ed.), Algal Symbiosis: A Continuum of ical convergence observed in Trentepohlia and Printzina is a general Interaction Strategies. Cambridge University Press, London and New York, pp. 173–204. trait of the evolution of subaerial green algae. This phenomenon Chapman, R.L., Henk, M.C., 1983. Ultrastructure of Cephaleuros virescens has been a great impediment in the systematics of these organisms (Chroolepidaceae, Chlorophyta). IV. Absolute configuration analysis of the 338 F. Rindi et al. / Molecular Phylogenetics and Evolution 52 (2009) 329–339
cruciate flagellar apparatus and multilayered structure in pre- and post-release López-Bautista, J.M., Rindi, F., Casamatta, D., 2007. The systematics of subaerial gametes. Am. J. Bot. 70, 1340–1355. algae. In: Seckbach, J. (Ed.), Algae and Cyanobacteria in Extreme Environments. Chapman, R.L., Waters, D.A., 2001. Lichenization of the Trentepohliales—complex Springer, Dordrecht, pp. 601–617. algae and odd relationships. In: Seckbach, J. (Ed.), Symbiosis. Kluwer Academic López-Bautista, J.M., Rindi, F., Guiry, M.D., 2006. Molecular systematics of the Publishers, Dordrecht, pp. 359–371. subaerial green algal order Trentepohliales: an assessment based on Cribb, A.B., 1970. A revision of some species of Trentepohlia especially from morphological and molecular data. Int. J. Syst. Evol. Microbiol. 56, 1709–1715. Queensland. Proc. R. Soc. Qld 82, 17–34. López-Bautista, J.M., Waters, D.A., Chapman, R.L., 2002. The Trentepohliales De Clerck, O., Leliaert, F., Verbruggen, H., Lane, C.E., De Paula, J.C., Payo, D.A., revisited. Constancea 83: http://ucjeps.berkeley.edu/constancea/83/lopezetal/ Coppejans, E., 2006. A revised classification of the Dictyoteae (Dictyotales, trentepohliales.html. Phaeophyceae) based on rbcL and 26S ribosomal DNA sequence analysis. J. López-Bautista, J.M., Waters, D.A., Chapman, R.L., 2003. Phragmoplastin, green algae Phycol. 42, 1271–1288. and the evolution of cytokinesis. Int. J. Syst. Evol. Microbiol. 53, 1715–1718. De Clerck, O., Verbruggen, H., Huisman, J.M., Faye, E.J., Leliaert, F., Schils, T., Maddison, D.R., Maddison, W.P., 2002. MacClade 4.05: Analysis of Phylogeny and Coppejans, E., 2008. Systematics and biogeography of the genus Pseudocodium Character Evolution. Sinauer Associates, Sunderland, MA. (Bryopsidales, Chlorophyta), including the description of P. Natalense sp. nov. von Martius, C.F.P., 1817. Flora Cryptogamica Erlangensis. J.L. Schrag, Nürnberg. from South Africa. Phycologia 47, 225–235. McCourt, R.M., Karol, K.G., Bell, J., Helm-Bychowski, M., Grajewska, A., De Wildeman, E., 1891. Les Trentepohlia des Indes Neerlandaises. Ann. Jard. Bot. Wojciechowski, M.F., Hoshaw, R.W., 2000. Phylogeny of the conjugating green Buitenzorg 9, 127–142. algae (Zygnemophyceae) based on rbcL sequences. J. Phycol. 36, 747–758. De Wildeman, E., 1900. Les algues de la flore de Buitenzorg. E.G. Brill, Leiden. Millardet, M.A., 1870. De la germination des zygospores dans les genres Closterium Ettl, H., Gärtner, G., 1995. Syllabus der Boden-, Luft- und Flechtenalgen. Gustav et Staurastrum et sur un genre nouveau d’algues chlorosporées. Mém. Soc. Sci. Fischer Verlag, Stuttgart, Jena and New York. Nat. Strasbourg 6, 37–51. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the Nakada, T., Nozaki, H., Pröschold, T., 2008. Molecular phylogeny, ultrastructure and bootstrap. Evolution 39, 783–791. taxonomic revision of Chlorogonium (Chlorophyta): emendation of Flint, E.A., 1959. The occurrence of zoospores in Physolinum Printz. New Phytol. 58, Chlorogonium and description of Gungnir gen. nov. and Rusalka gen. nov.. J. 267–270. Phycol. 44, 751–760. Gontcharov, A.A., Marin, B., Melkonian, M., 2004. Are combined analyses better than Nakano, T., Handa, S., 1984. Observations on Trentepohlia lagenifera (Hild.) Wille single gene phylogenies? A case study using SSU rDNA and rbcL sequences (Chlorophyceae, Trentepohliaceae). Jpn. J. Phycol. 32, 354–363. comparisons in the Zygnematophyceae (Streptophyta). Mol. Biol. Evol. 21, 612– Nakano, T., Ihda, T., 1996. The identity of photobionts from the lichen Pyrenula 624. japonica. Lichenologist 28, 437–442. Hansgirg, A., 1886. Prodromus der algenflora von Böhmen. Erster theil enthaltend Nozaki, H., Misawa, K., Kajita, T., Kato, M., Nohara, S., Watanabe, M.M., 2000. Origin die Rhodophyceen, Phaeophyceen und einen theil der Chlorophyceen. Arch. and evolution of the colonial Volvocales (Chlorophyceae) as inferred from Naturwiss. Landesdurchf. Böhmen 5, 1–96. multiple chloroplast gene sequences. Mol. Phylogenet. Evol. 17, 256–268. Hariot, P., 1889a. Notes sur le genre Trentepohlia Martius (Suite). J. Bot. Paris 3, 378– Nylander, W., 1862. Quelques observations sur le genre Coenogonium. Ann. Sci. Nat. 388. Bot. ser. 4 16, 83–96. Hariot, P., 1889b. Notes sur le genre Trentepohlia Martius (Suite). J. Bot. Paris 3, 366– Olsen, J.L., Stam, W.T., Berger, S., Menzel, D., 1994. 18S rDNA and evolution in the 375. Dasycladales (Chlorophyta): modern living fossils. J. Phycol. 30, 729–744. Hariot, P., 1890. Notes sur le genre Trentepohlia Martius (Suite). J. Bot. Paris 4, 178– Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution. 180. Bioinformatics 14, 817–818. Hayden, H.S., Waaland, J.R., 2002. Phylogenetic systematics of the Ulvaceae (Ulvales, Printz, H., 1921. Subaerial algae from South Africa. Kong. Norsk. Vidensk. Selsk. Ulvophyceae) using chloroplast and nuclear DNA sequences. J. Phycol. 38, Skrift. 1, 3–41. 1200–1212. Printz, H., 1939. Vorarbeiten zu einer monographie der Trentepohliaceen. Nytt Mag. Hepperle, D., Nozaki, H., Hoenberger, S., Huss, V.A.R., Morita, E., Krienitz, L., 1998. Naturvbidensk. 80, 137–210. Phylogenetic position of the Phacotaceae within the Chlamydophyceae as Pröschold, T., Leliaert, F., 2007. Systematics of the green algae: conflict of classic and revealed by analysis of 18S rDNA and rbcL sequences. J. Mol. Evol. 47, 420–430. modern approaches. In: Brodie, J., Lewis, J. (Eds.), Unravelling the Algae: The Hildebrand, F., 1861. Über einen Chroolepus mit Zoosporenbildung. Bot. Zeit. 13, 81– Past, Present and Future of Algal Systematics. The Systematics Association 85. Special Volume Series 75. CRC Press, Boca Raton, London and New York, pp. Hillis, D.M., Huelsenbeck, J.P., 1992. Signal, noise and reliability in molecular 123–153. phylogenetic analyses. J. Hered. 83, 189–195. Rausch, H., Larsen, N., Schmitt, R., 1989. Phylogenetic relationships of the green Howland, L.J., 1929. The moisture relations of terrestrial algae. IV. Periodic algae Volvox carteri deduced from small-subunit ribosomal RNA comparisons. J. observations of Trentepohlia aurea Martius. Ann. Bot. 43, 173–202. Mol. Evol. 29, 255–265. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogeny. Rindi, F., Guiry, M.D., 2002. Diversity, life history and ecology of Trentepohlia and Bioinformatics 17, 754–755. Printzina (Trentepohliales, Chlorophyta) in urban habitats in western Ireland. J. Huss, V.A.R., Frank, C., Hartmann, E.C., Hirmer, M., Kloboucek, A., Seidel, B.M., Phycol. 38, 39–54. Wenzeler, P., Kessler, E., 1999. Biochemical taxonomy and molecular phylogeny Rindi, F., Lam, D.W., López-Bautista, J.M., 2008a. Trentepohliales (Ulvophyceae, of the genus Chlorella sensu lato (Chlorophyta). J. Phycol. 35, 587–598. Chlorophyta) from Panama. Nova Hedwigia 87, 421–444. Jobb, G., von Haeseler, A., Strimmer, K., 2004. TREEFINDER: a powerful graphical Rindi, F., Guiry, M.D., López-Bautista, J.M., 2008b. Distribution, morphology and analysis environment for molecular phylogenetics. 2004. BMC Evol. Biol. phylogeny of Klebsormidium (Klebsormidiales, Charophyceae) in urban Available from:
Starr, R.C., Zeikus, J.A., 1987. UTEX—the culture collection of algae at the University Verbruggen, H., Theriot, E.C., 2008. Building trees of algae: some advances in of Texas at Austin. J. Phycol. 23 (Suppl.), 1–47. phylogenetic and evolutionary analysis. Eur. J. Phycol. 43, 229–252. Swofford, D.L., 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Wakasugi, T., Nagai, T., Kapoor, M., Sugita, M., Ito, M., Ito, S., Tsudzuki, J., Nakashima, Methods). Version 4.0b10. Sinauer Associates, Sunderland, MA. K., Tsudzuki, T., Suzuki, Y., Hamada, A., Ohta, T., Inamura, A., Yoshinaga, K., Thompson, R.H., Wujek, D.E., 1992. Printzina gen. nov. (Trentepohliaceae), including Sugiura, M., 1997. Complete nucleotide sequence of the chloroplast genome a description of a new species. J. Phycol. 28, 232–237. from the green alga Chlorella vulgaris: the existence of genes possibly involved Thompson, R.H., Wujek, D.E., 1997. Trentepohliales: Cephaleuros, Phycopeltis and in chloroplast division. Proc. Natl. Acad. Sci. USA 94, 5967–5972. Stomatochroon. Morphology, Taxonomy and Ecology. Science Publishers, Watanabe, S., Mitsui, K., Nakayama, T., Inouye, I., 2006. Phylogenetic relationships Enfield, New Hampshire. and taxonomy of sarcinoid green algae: Chlorosarcinopsis, Desmotetra, Tompkins, J., Deville, M.M., Day, J.G., Turner, M.F., 1995. Culture collection of algae Sarcinochlamys, gen. nov., Neochlorosarcina and Chlorosphaeropsis and protozoa. Catalogue of strains. The Culture Collection of Algae and Protozoa, (Chlorophyceae, Chlorophyta). J. Phycol. 42, 679–695. Institute of Freshwater Ecology, Ambleside. Xia, X., Xie, Z., 2001. DAMBE: data analysis in molecular biology and evolution. J. Van den Hoek, C., Mann, D.G., Jahns, H.M., 1995. Algae: An Introduction to Hered. 92, 371–373. Phycology. Cambridge University Press, Cambridge. Xia, X., Xie, Z., Salemi, M., Chen, L., Wang, Y., 2003. An index of substitution Verbruggen, H., Littler, D.S., Littler, M.M., 2007a. Halimeda pygmaea and Halimeda saturation and its application. Mol. Phylogenet. Evol. 26, 1–7. pumila (Bryopsidales, Chlorophyta): two new dwarf species from fore reef Zechman, F.W., 2003. Phylogeny of the Dasycladales (Chlorophyta, Ulvophyceae) slopes in Fiji and the Bahamas. Phycologia 46, 513–520. based on analyses of Rubisco large subunit (rbcL) gene sequences. J. Phycol. 39, Verbruggen, H., Leliaert, F., Maggs, C.A., Shimada, S., Schils, T., Provan, J., Booth, D., 819–827. Murphy, S., De Clerck, O., Littler, D.S., Littler, M.M., Coppejans, E., 2007b. Species Zeller, G., 1873. Algae collected by Mr. S. Kurz in Arracan and British Burma, boundaries and phylogenetic relationships within the green algal genus Codium determined and systematically arranged. J. Asiat. Soc. Bengal 42, (Bryopsidales) based on plastid DNA sequences. Mol. Phylogenet. Evol. 44, 240–254. 175–193.