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Endophytic Xylariaceae: diversity and taxonomy inferred from rDNA sequence analyses

Endophytic Xylariaceae: diversity and taxonomy inferred from rDNA sequence analyses


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"Thailand is considered as one of the areas containing a high percentage of unknown taxa of Xylariaceae (Rogers 2000). In Thailand, several studies on endophytic fungi have been documented, namely, from several bamboos (Luyong et al. 2000), banana (Musa acuminata) (Photita et al. 2001), monkeypod/rain tree (Samanea saman) (Chareprasert et al. 2006), and several xylariaceous endophytes were also reported from those plants."

Review quoted from Izumi okane, Prasert Srikitikulchai, Kyoko Toyama, Thomas Laessoe etc. Full Paper; Study of endophytic Xylariaceae in Thailand: diversity and taxonomy inferred from rDNA sequence analyses with saprobes forming fruit bodies in the field.

Have any of you known the similar cases (the high percentage of Xylariaceae) in the USA, France, or UK, proved by reference(s)?


Nuclear ribosomal 18S and internal transcribed spacer (ITS) sequence data were used to identify endophytic fungi cultured from six species of liverworts collected in Jamaica and North Carolina. Comparisons with other published fungal sequences and phylogenetic analyses yielded the following conclusions: (1) the endophytes belong to the ascomycete families Xylariaceae, Hypocreaceae, and Ophiostomataceae, and (2) liverwort endophytes in the genus Xylaria are closely related to each other and to endophytes isolated from angiosperms in China, Puerto Rico, and Europe. Liverwort endophytes are expected to be foragers or endophytic specialists, although little is known about the role of these fungi in symbioses. Features that may indicate a mutualistic role for these endophytes are discussed.

Endophytic fungi that live inside healthy plant tissue without apparent damage to the host are found in all lineages of land plants (Petrini and Petrini, 1985). New species are continually being described from cultural and molecular studies of plant tissue, and endophyte biology is a burgeoning field in mycology. (A Biological Abstracts search for “endophyt*” in the title retrieved 923 papers from 1990 through January 2003.) These studies indicate that the breadth of endophyte diversity and ecology is just beginning to be discovered (Arnold et al., 2000).

Many groups of fungi exist as endophytes, though most are ascomycetes. Well-known examples are Clavicipitaceae (e.g., Epichloe) species that inhabit grasses (Poaceae). Endophytic associations with Epichloe have been shown to be mutualistic: the plant receives protection from herbivory through fungal toxins, and the fungus receives host tissue as a nutritive source, along with seed-mediated dispersal of mycelia (reviewed in Clay, 1988). However, the ecology and distribution of most groups of endophytic fungi remain poorly known.

Endophytic Xylariaceae have been documented in conifers, monocots, dicots, ferns, and lycopsids (Brunner and Petrini, 1992). One hypothesis for the role of Xylariaceae endophytes holds that the fungus is a quiescent colonizer and will later decompose cellulose and lignin when the plant begins to senesce (Petrini et al., 1995 Whalley, 1996). However, growing evidence suggests that some xylariaceous fungi may exist solely as endophytes (Rogers, 2000 J. D. Rogers, Washington State University, personal communication). No obvious benefit to living host plants has been documented for Xylariaceae.

Liverworts are nonvascular, spore-bearing plants, or “bryophytes.” Though these plants have long been known to form associations with fungi (see Boullard [1988] and Read et al. [2000] for review), few liverwort endophytes have been identified with certainty. Duckett and Read (1995) grew ascomycetes from 11 British liverworts and through cross-inoculation experiments with angiosperms concluded that the fungi were likely Hymenoscyphus ericae (D. J. Read) Korf and Kernan (Leotiaceae), the ascomycete that forms mycorrhizae with the flowering plant family Ericaceae. This species was also identified from an Antarctic liverwort [Cephaloziella exilifora (Taylor) Stephani (Cephaloziellaceae)] based on DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) (Chambers et al., 1999). It is unclear whether Xylariaceous endophytes previously isolated from “bryophytes,” as listed in Petrini and Petrini (1985), included any liverworts. Endophytes of some liverwort species are restricted to the rhizoids, while those of other liverwort species can be detected growing within the thallus. Most rhizoid-associated endophytes are thought to be ascomycetes, while those within thalli are thought to be basidiomycetes or Glomalean fungi (Boullard, 1988). The resemblance of these associations to vascular plant mycorrhizae have led some to label them as mutualistic, though the nature of the symbiosis remains poorly understood (Read et al., 2000).

The goal of this study was to characterize the endophytic communities of six common liverworts collected in Jamaica and North Carolina, USA. The study consisted of three parts: (1) morphological observations of the fungal infection, (2) identification of the endophytes based on nrDNA similarity and phylogeny, and (3) ecological comparisons of the endophytes with related fungal species.


Endophytic Xylariaceae: diversity and taxonomy inferred from rDNA sequence analyses - Biology

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Vamsapriya (Xylariaceae) Re-Described, with Two New Species and Molecular Sequence Data

Dong-Qin Dai, 1,2,3 Ali H. Bahkali, 4 Qi-Rui Li, 5 D. Jayarama Bhat, 6,7 Nalin N. Wijayawardene, 1,6 Wen-Jing Li, 1,6 Ekachai Chukeatirote, 1,6 Rui-Lin Zhao, 8 Jian-Chu Xu, 2,3 Kevin D. Hyde 1,4,2,*


Results

Across the diverse biomes and plant taxa surveyed here, culturable fungi from living leaves were isolated less frequently and were less diverse than those isolated from non-living leaves. Fungal communities in living leaves also differed detectably in composition from communities in dead leaves and leaf litter within focal sites and host taxa, regardless of differential weighting of rare and abundant fungi. All focal isolates grew on cellulose, lignin, and pectin as sole carbon sources, but none displayed ligninolytic or pectinolytic activity in vitro. Cellulolytic activity differed among fungal classes. Within Dothideomycetes, activity differed significantly between fungi from living vs. non-living leaves, but such differences were not observed in Sordariomycetes.


Summary

  • The initial submission of this article was received on April 17th, 2016 and was peer-reviewed by 3 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on April 29th, 2016.
  • The first revision was submitted on September 29th, 2016 and was reviewed by 2 reviewers and the Academic Editor.
  • A further revision was submitted on November 6th, 2016 and was reviewed by the Academic Editor.
  • The article was Accepted by the Academic Editor on November 7th, 2016.

Endophytic Xylariaceae: diversity and taxonomy inferred from rDNA sequence analyses - Biology

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Endophytic Diaporthe from Southeast China are genetically diverse based on multi-locus phylogeny analyses

Species of Diaporthe (anamorph Phomopsis) comprise a diverse and widely distributed group of phytopathogens, saprophytes and endophytes. However, the degree of genetic diversity of endophytic Diaporthe has not yet been fully investigated. In this paper, a survey of endophytes from 28 plants in southeast China yielded 116 Diaporthe isolates, out of which 64 haplotypes were determined using DnaSP ver. 5.1 based on alignment result of internal transcribed spacer of ribosomal nucleotide sequences (ITS rDNA). Many haplotypes turned out to be quite different from known species and displayed high diversity. Among them, 14 strains from 5 discriminating terminal clades were selected to go through further analysis according to partial sequence of translation elongation factor 1-&alpha (tef1-&alpha) and again they got separated from others. The following multi-gene phylogenetic analysis based on ITS rDNA, tef1-&alpha, &beta tubulin and calmodulin grouped eight most discrepant strains into three distinctive clusters, cluster 1 (Rc001, Eu004 and Eu009), cluster 2 (ZJWCF252, Sjm001 and Ac001) and cluster 3 (Pcs013 and Sfp005) respectively with high support values. These clusters above represent three potentially novel species. This research provides strong evidence of high biodiversity and novelty of Diaporthe endophytes from southeast China, which is thus important not only for better resolving the taxonomy in this genus, but also for further utilization due to their multiple application.

Journal

World Journal of Microbiology and Biotechnology &ndash Springer Journals


Taxonomy

Neotyphodium sibiricum sp. nov. X. Zhang et Y.B. Gao ( Fig. 1a and b)

Colonia in PDA tarde crescens, aetate 4 hebdomadum in temperatura 25C culta diametrum 9–12 mm attingens. Coloniae ex superficie agari elevatae, non vel subconvolutae, hyphis aeriis in massas albas gossypinas dispositis, in parte aversa bubalinae vel pallide brunneae, agaro interdum frangenti. Hyphae vegetativae hyalinae, septatae, 1.3–2.6 μm latae, in interstitiis inter cellulas vaginae foliaris et medulla caulis reproductivi et testa seminum intima inventae. Cellulae conidiogenae hyalinae, simplices, orthotropicae, ut prolationes laterales hypharum aeriarum ortae, saepe prolificantes, 24.1–40.5 μm longae, ad basem 1.6–2.2 μm latae, sensim apicem versus contractae usque ad 0.8–1.0 μm latae, ad quidque locum conidiogenum conidio unico evoluto. Conidia hyalina, laevia, aseptata, ellipsoidea, navicularia vel lunata, 3.0–4.6 × 1.7–2.2 μm. Sporulatio in cultura copiosa. Teleomorpha ignota. Neotyphodio guerinii J.-J. Guillaumin, C. Ravel et C.D. Moon, N. gensuensi C.J. Li et Z.B. Nan, et N. gansuensi var. inebrianti C.D. Moon et C.L. Schardl. genetice similis.

Colonies on PDA slow-growing, reaching a diameter of 9–12 mm after 4 weeks at 25 °C ( Fig. 2a). Colonies raised from agar surface, not convoluted or slightly convoluted, white, cottony, mass of aerial hyphae. Colony reverse tan to pale brown, fracturing of agar occurs. Vegetative hyphae hyaline, septate, 1.3–2.6 μm wide, in the intercellular of leaf sheath, pith of the reproductive stem and innermost seed coat. Conidiogenous cells hyaline, unbranched, orthotropic, arising as lateral extensions of the aerial hyphae, frequently proliferating, 24.1–40.5-μm long, 1.6–2.2-μm wide at the base, gradually tapering to 0.8–1.0-μm wide at the apex, producing just one conidium at each conidiogenous locus. Conidia hyaline, smooth, aseptate, ellipsoid, navicular to lunate, 3.0–4.6 × 1.7–2.2 μm ( Fig. 2b). Sporulation in culture abundant. Teleomorph unknown. Shows genetic similarity to one of two haplotypes of N. guerinii J.-J. Guillaumin, C. Ravel et C.D. Moon, N. gansuense C.J. Li et Z.B. Nan and N. gansuense var. inebrians C.D. Moon et C.L. Schardl.

Etymology: The endophyte is named referring to the host species.

Holotype: Isolated from seeds of A. sibiricum originally collected in IMGERS-CAS in Inner Mongolia, China, in September 2006 by X. Zhang, deposited in Mycological Herbarium of Nankai University, China (MHNU-ASIB02).


Discussion

The highly diverse nonclavicipitaceous endophytes can be divided into 3 functional groups based upon host colonization patterns, mechanism of transmission, biodiversity levels within plant tissues and ecological function [19]. All classes have broad host ranges but class 3 exist as highly localized independent infections restricted to above ground plant tissues allowing for extremely high in planta biodiversity while class 4 are primarily ascomycetous fungi restricted to roots where they form inter- and intracellular hyphae and microsclerotia and are capable of extensive tissue colonization [19]. Members of class 2, species within the Ascomycota or Basidiomycota such as the fungi we have isolated from leaves of Sarracenia species, infect and colonize via appressoria or by direct penetration by hyphae [25]. Some are known to confer fitness benefits to their host, especially if there are habitat-specific selective pressures placed on the host [26]. These selective pressures have been associated with pH, temperature, drought, and salinity but also likely include growth in nutrient-deficient soils. Class 2 endophytes have also been demonstrated to increase plant biomass under stressful conditions [19]. Colonization of Sarracenia plants by endophytes in the spring may account for the subsequent increase in pitcher size observed over the course of the growing season particularly for S. leucophylla. Many endophytes produce phytohormones such as indole-3-acetic acid, cytokines, and other plant growth-promoting substances [27]. We also speculate that endophyte-infected plants benefit from their fungal associates by their influence on nutrient availability from within pitchers and, possibly, by directly influencing the biota within the pitchers. Tan and Zou [27] postulate that horizontal gene transfer may explain why some endophytes are capable of producing phytochemicals characteristic of the host. Although speculative at this point, carnivorous host plants may have co-evolved along with fungi and now rely on endophytes for production or augmentation of levels of some of the digestive enzymes found within their pitchers.

Cost-benefit models predict that carnivory could result in an increased rate of photosynthesis manifested in one form as an increase in total leaf mass, but experiments to support this have been equivocal [13], [28], [29]. While most nutrient supplementation studies do identify a significant increase in growth, excess nutrients may not lead directly to increased photosynthetic rates [8]. Cyperus erythrorhizos Muhl. (Redroot Flatsedge), a C4 plant growing in nutrient deficient wetlands, habitat similar to that for many Sarracenia, have a high photosynthetic nitrogen use efficiency (PNUE) [30] in contrast to the low PNUE of Sarracenia [13], which are likely C3 plants. The activity of endophytes within pitcher plant leaf tissue may explain the PNUE observations in comparison to non-carnivorous plants growing in similar habitats. The presence of endophytes could also help discern why discordant data exists between the construction costs of carnivorous leaves versus phyllodia, help elucidate how carbon derived from prey is utilized, and further clarify the relationship between plant biomass and photosynthesis in nutrient-manipulated plants. For example, non-feeding Sarracenia are phosphorus-limited or nitrogen+phosphorus co-limited while artificially fed plants are more strongly nitrogen-limited [13]. Of the nitrogen available from prey, 60% is sequestered by bacteria in the pitchers [4], [31]. Over a growing season, plants will rely on prey-based phosphorus to boost photosynthetic efficiency due to nitrogen-limitation from bacterial sequestration [13]. It has been observed that endophyte growth limitation by lack of an adequate nitrogen source or phosphate components of the nutritional environment leads to the synthesis of secondary metabolites [32] and these metabolites, released into the plant tissue or pitcher milieu, can alter growth characteristics if they are plant growth regulators or impact the micro-organisms present within the pitcher if antimicrobial in nature.

The endophyte isolates of Sarracenia may contribute to their hosts' fitness by means of the production of biologically active compounds. Basidiomycetous endophytes are rarely isolated from higher plants and these are often orchid mycorrhizas [33]. While many of these latter species are associated with white rot and brown rot of trees and may be saprobes or latent pathogens, their production of lignocellulolytic enzymes and potential to produce bioactive secondary metabolites may favor their association with Sarracenia pitchers. S. purpurea, for example, from which two basidiomycete isolates were obtained, has open pitchers that often accumulate plant debris. Degradation of captured plant debris by these fungi would be beneficial to host and endophyte.

Every Ascomycota endophyte taxa isolated from Sarracenia in this study has been proven to produce biologically active compounds. Two isolates representative of the Pleosporales were found, one being a member of the polyphyletic anamorph Paraconiothyrium/Coniothyrium and the other being an unidentified taxon. Members of the Pleosporales exhibit a diversity of habits, including parasites, saprobes, and endophytes [34]. Preussomerins, isolated from various fungi, including Edenia gomezpompae M.C. González, Anaya, Glenn, Saucedo, and Hanlin, a Pleosporales endophyte of Callicarpa, possess a wide range of biological properties, including antibacterial, algicidal, herbicidal, antiplasmodial, antitumor, and antifungal activities, including inhibition of other endophytes and phytopathogens [35]. Interestingly, the genus Paraconiothyrium was erected to segregate mycoparasitic species from Coniothyrium species [36]. An endophyte that utilized other fungi as a food source could influence competition for resources within the flooded and enzyme-poor pitchers of S. purpurea, from which this isolate was made.

Endophytic penicillia are widespread and heterogenous, and various species have been reported as endophytes of plants with 20 species of Penicillium isolated from the roots of Picea mariana (Miller) Britton Sterns, and Poggenburg (Black Spruce) alone [37]. By the end of 1984, over 380 biologically active metabolites were known from Penicillium [32] and even more are now known. Of the 6450 biologically active compounds identified from microfungi up through 2009, over 30% have been obtained from Aspergillus and Penicillium [38] and these compounds exhibit a vast diversity of activity.

Cryptosporiopsis spp. (teleomorph Pezicula spp.) have had biologically active compounds isolated that exhibit antibacterial, antifungal and algicidal activities [39]. Echinocandin isolated from Cryptosporiopsis sp. and Pezicula sp. was shown, in vitro, to inhibit pathogens of the respective host plants [40]. Many strains of Pezicula synthesize, in vitro, fungicidal compounds such as mullein, mycorrhizin, epi-ethiosolide, cryptosporopsin, and cryptocandin [39], [41] postulated to help it to maintain a mutualistic role with its host.

Potent antifungal agents, the sordarins, are synthesized by members of the Xylariales, a trait more frequently attributed to this group than to any other fungal order [42]. Species of Xylaria have been shown to produce potent antifungal and antibacterial metabolites [43]. The Xylariales may either be colonizers that will later decompose cellulose when the host plant dies or be true endophytes, although obvious benefits to the host plant have yet to be documented [44].

Phomopsis and Colletotrichum are common isolates from dicot leaves and frequently dominate endophyte assemblages of the host [45]. Phomopsis spp. are not host specific and exhibit high host variability. The genus is a very rich source of secondary biological compounds with antifungal, herbicidal, algicidal, antimicrobial, and plant growth regulating activities [46].

Fungi of the genus Colletotrichum are well documented as significant plant endophytes [47]–[50]. C. gloeosporioides in particular has been reported from at least 470 host genera and more than 2000 taxa [51] primarily as a host-specific pathogen although C. gloeosporioides is now recognized as a broadly defined species complex that contains multiple clades, each of which may represent a genetically isolated species [52]. Lu et al. [50] isolated metabolites of a Colletotrichum sp. from Artemisia annua and Zou et al. [23] similarly isolated metabolites of C. gloeosporioides from Artemisia mongolica that have antimicrobial and antifungal properties. Tan and Zou [27] found that culture broth from C. gloeosporioides could promote the growth of host callus. As this genus was represented in every species of Sarracenia studied, and isolated multiple times, there is great potential for a mutualistic relationship between this endophyte and host that could be further elucidated by an examination of the secondary products, if any are produced by the fungus and compared to the native metabolites and enzymes produced by sterile plants. Furthermore, some light could be shed on the problem of nutrient limitation and stoichiometry of carnivorous plants should sterile and endophyte-infected plants be utilized and compared under experimental conditions.

It is well established that some of the large number of metabolites produced by endophytes offer a significant benefit to their host plant [27]. These benefits could contribute to the protection and survival of the host by acting as growth regulators, antimicrobials, and mediators of environmental stress. In the case of Sarracenia, these metabolites could have an even broader impact due to the unique structure of the trap leaf, which would function to capture and store excreted materials within the pitcher where the chemicals would interact with prey as well as a complex microcosm of associated organisms.

This is the first instance of Coniothyrium/Paraconiothyrium, Penicillium, Cryptosporiopsis, Phomopsis and Colletotrichum spp. ascertained to be endophytes of the family Sarraceniaceae and, to our knowledge, the first report of fungal endophytes of leaves of any carnivorous and proto-carnivorous plant family. The isolation of Colletotrichum spp. from multiple Sarracenia individuals of all 4 species at locations 300+ miles apart, as well as during different years, strongly suggests that at least this fungal genus is a true pitcher plant endophyte. This study only concerned the isolation of fungal endophytes. Our preliminary assay also indicates a potential diversity of bacterial endophytes as well. The role, if any, of the fungal endophytes we isolated from Sarracenia is unknown as is whether they produce any biological compounds that may be of benefit to the plants. At this time it is unknown if these endophytes truly contribute to carnivory in Sarracenia and further investigation concerning the role of endophytes and/or their metabolites in successful carnivory is highly desired.


Discussion

To exclude incongruent taxon sampling and likely artifacts associated with it, the two genes (rbcL and SSU rDNA) were sequenced exclusively from the same strain. The importance of this strict approach is shown by two examples: (1) two strains designated Netrium oblongum (M 1367 and SVCK 255 in morphology both corresponding to the species description) are in fact unrelated to each other, and (2) a database sequence (U38694) of strain UTEX 934 (Roya anglica) apparently was not derived from this strain, but refers to another genus, as shown in this study. Combining SSU rDNA and rbcL sequences of different strains may thus introduce taxon sampling artifacts, which could result in chimeric sequences and in single-gene trees in conflicting topologies. Our approach ensures that conflicts between single-gene topologies are derived from different patterns of sequence evolution between the genes.

SSU rDNA and rbcL sequence comparisons of 43 strains of the Zygnematophyceae were used to analyze the relation between single-gene verses combined analyses, gene concatenation versus log-likelihood summation, and bootstrap percentages (ML, NJ, and MP) versus posterior probabilities (BI), as an evolutionary case study based on empirical data. Previously published phylogenetic analyses using SSU rDNA sequence comparisons in the Zygnematophyceae suffered mostly from lack of resolution ( Besendahl and Bhattacharya 1999 Denboh, Hendrayanti, and Ichimura 2001 Gontcharov, Marin, and Melkonian 2003), whereas in rbcL studies, taxon sampling was limited, with only one species per genus included ( McCourt et al. 2000). In phylogenetic analyses of streptophyte green algae, different molecular markers favored conflicting tree topologies for example., concerning the position of the genera Mesostigma, Klebsormidium, or the group studied here, the Zygnematophyceae ( Marin and Melkonian 1999 Lemieux, Otis, and Turmel 2000 McCourt et al. 2000 Karol et al. 2001 Cimino and Delwiche 2002 Delwiche et al. 2002 Martin et al. 2002 Turmel et al. 2002a).

Single-Gene Data and Analyses

In the Zygnematophyceae, ribosomal (SSU rDNA) and chloroplast (rbcL) genes studied revealed considerable differences in their evolutionary dynamics as reflected by model parameters (patterns of nucleotide substitutions, Γ, base composition). In both genes, the C↔T substitution category is conspicuously high in comparison with the remaining frequencies ( table 3), a situation, which for SSU rDNA may be related to pairing constraints at the transcript (rRNA) level (G-C↔G-U) and for rbcL (an even higher C↔T value) is caused by asymmetrical codon usage ( Morton 1994). The gamma-shaped parameter estimated for the SSU rDNA data set is nearly three-fold lower than that for rbcL, in which the variability distribution reflects the regular codon structure. Moreover, the length of the Rubisco large subunit is conserved in the Viridiplantae (476 amino acids), and the variability at the amino acid level is rather moderate (reviewed by Kellogg and Juliano [1997]). In the Zygnematophyceae, less than 80% of the sequence variability of rbcL refers to third codon positions. Although third codon positions are sometimes down-weighted or excluded from the phylogenetic analyses because of codon degeneration and homoplasy ( Nickrent et al. 2000 Nozaki et al. 2000), restriction of our analyses to first and second codon positions resulted in greatly diminished resolution (see also McCourt et al. [2000]). However, using only the third codon position, the topology and resolution reflected that obtained for all positions, thus, demonstrating that most of the phylogenetic signal resides in the third codon.

Not surprisingly, SSU rDNA and rbcL data led to selection of different models of evolution and model parameters ( table 3), but these models were not in conflict (TrN is nested within GTR) and do not prevent a combined analysis using a concatenated (averaged) model (here: GTR).

In general, both single-gene trees show a high degree of congruence and largely recover the same taxa and clades with comparable BP and PP support. However, the analyses also reveal some conflicts that are not caused by incongruence in taxon sampling. The most obvious discrepancy between the two single-gene trees relates to the split between the orders Zygnematales and Desmidiales, which is resolved in the SSU rDNA analysis but not in rbcL phylogenies ( McCourt et al. 2000 this study) in our rbcL study, the Desmidiales are mixed with two zygnematalean branches (Roya and Netrium oblongum SVCK 255). However, even this incongruence refers to internal branches without significant bootstrap support in the rbcL analysis (in contrast to PP [see below]), and moving Roya and N. oblongum SVCK 255 to the Zygnematales is not rejected in user-defined topology tests ( table 4). As an example for congruence, the polyphyly of Mesotaenium, Cylindrocystis, and Netrium is clearly revealed in both single-gene phylogenies.

Combined Analyses

When both data sets were combined as a concatenated “supergene” and analyzed with a single average model, the resulting phylogeny was superior to both single-gene analyses when the statistical support of internal branches is considered. Specifically, concatenated data significantly (BP and PP) resolved the major conflict between rbcL and SSU rDNA trees (Desmidiales-Zygnematales divergence) in favor of the SSU rDNA analysis (i.e., monophyly of both groups in the unrooted phylogeny). Several other internal branches (especially basal branches of the Zygnematales), which in single-gene phylogenies were not or weakly supported, obtained higher significance in the combined analysis (see Results). In general, the combined analysis was dominated by the phylogenetic signal of the SSU rDNA data set, whereas rbcL contributed sufficient sequence diversity to improve resolution within clades (see MZC clade where SSU rDNAs are almost identical) but also to increase the overall significance of the branches.

It is somewhat illegitimate (a logical circle) to use higher statistical support for branches as the only criterion to regard combined analysis as superior (i.e., closer to the “true tree” than the rbcL analysis). Of course, there is no a priori knowledge of the “true tree.” However, cell-wall characters provide some independent evidence for comparing evolutionary hypotheses in the Zygnematales. The SSU rDNA tree reveals a single evolutionary transition from simple cell walls (Zygnematales) towards complex cell walls in the monophyletic Desmidiales, without homoplasious character changes. The conflicting rbcL scenario, if correct, would imply two additional changes (i.e., reversals: complex→simple wall) for those Zygnematales that are rooted within the Desmidiales (Roya and Netrium oblongum SVCK 255). Thus, the parsimony criterion applied to cell-wall structures (one versus three character changes), as well as the combined SSU rDNA+rbcL analyses, both favor the SSU rDNA topology and emphasize the value of complex cell walls as a phylogenetic marker.

In a concatenated analysis, it is possible that the longer or the more variable data set dominates the “average” model of sequence evolution and, thus, the resulting topology. However, the LS method applied here (which avoids the use of an average model) agreed with the results obtained by the concatenated analysis. There are, of course, conditions under which concatenated phylogenetic analyses will fail. Whenever different genes evolve under deviating rules (i.e., models), a concatenated analysis may be significantly worse than phylogenies that allow the application of separate models, as shown by Pupko et al. (2002). In the Zygnematales, the two single-gene models were not in conflict (see above), but some model parameters, especially the gamma-shaped and the C↔T substitution category, differed considerably between SSU rDNA and rbcL (see Results). The concatenated model (averaged parameters) apparently did not violate the evolutionary characteristics of both genes.

Combined Analyses and Long Branches

It has been proposed that fast-evolving taxa with long undivided branches should be better analyzed by using another, slow-evolving, gene to avoid LBA ( Philippe 2000). Fortunately, all long-branch taxa in our analyses are either fast-evolving in rbcL (M. endlicherianum and N. oblongum SVCK 255) or in the SSU rRNA gene (Spirogyra) but not in both data sets. Our results show that the combination of slow-evolving and fast-evolving genes can also resolve the phylogenetic position of a taxon with a fast-evolving gene, in particular when improved taxon sampling does not help to subdivide the long branch (e.g., SSU rDNA in Spirogyra). It appears that the combined approach can successfully extract the phylogenetic signal from the fast-evolving gene and nevertheless reduce LBA (see also Hoef-Emden, Marin, and Melkonian [2002]). The latter is illustrated by comparison of the positions of Mesotaenium endlicherianum and Netrium oblongum SVCK 255 in single-gene and combined analyses. Both taxa have long branches in the rbcL phylogeny but not in the SSU rDNA phylogeny. Their position in the rbcL tree ( fig. 2) most likely reflects a LBA artifact because in combined analyses, they comprise shorter (albeit still relatively long) branches (no LBA), and their placement confirms the SSU rDNA topology with better significance. The most conspicuous long branch in our analyses refers to the genus Spirogyra in SSU rDNA trees, in which Spirogyra was previously positioned as a sister to all other Zygnematophyceae ( Besendahl and Bhattacharya 1999) or as one of the basal divergences of the class ( Gontcharov, Marin, and Melkonian 2003). In the rbcL phylogeny, Spirogyra is not a long-branch taxon, but interestingly, the position of Spirogyra does not contradict rooted ( Gontcharov, Marin, and Melkonian 2003) or unrooted (this study) SSU rDNA trees. We conclude that the position of Spirogyra in the SSU rDNA trees was likely not the result of an LBA artifact.

The examples discussed above may provide some confidence that the deep-level phylogeny of the Zygnematophyceae based on the conservative SSU rRNA gene agrees better with organismal data and is less sensitive to various artifacts than analyses using the homoplasious and largely saturated gene rbcL. However, the latter gene resolves shallow evolutionary relationships much better where the conserved SSU rDNA lacks variability.

Phylogeny of the Zygnematophyceae

Although the Zygnematales, characterized by a simple cell wall (consisting of only one piece, no pores [ Mix 1972] plesiomorphic character state), form a clade in the unrooted trees, this taxon is not monophyletic because the root of the Zygnematophyceae falls within the Zygnematales, and, thus, reveals this order as paraphyletic ( McCourt et al. 2000 Gontcharov, Marin, and Melkonian 2003). Chloroplast shape (three types) and level of organization (unicellular or filamentous) vary in the Zygnematales and have previously been used for classification (e.g., Palla 1894 Randhawa 1959 Yamagishi 1963). Generally, none of these proposals gains support by our molecular phylogenetic analyses. We have tentatively identified seven lineages within the traditional Zygnematales, namely Roya, N, SPI, the “crown-Zygnematales,” and three individual taxa (Mesotaenium endlicherianum, Netrium interruptum, and N. oblongum SVCK 255). Three filamentous genera (Spirogyra, Mougeotia, Zygnema) are resolved as monophyletic, whereas other genera are not monophyletic. Obviously, in the Zygnematales, the genetic diversity at the genus level was severely underestimated by traditional taxonomists because the importance of the organizational level (unicellular versus filamentous) and chloroplast shape (axial platelike, stellate, or helical) has been overestimated. These characters may have originated or have been lost several times independently in the group. Our phylogenies place unicellular organisms as basal divergences of some zygnematalean clades (“crown-Zygnematales,” MZC, and MOUG) tentatively suggesting that unicells could perhaps be ancestral to these lineages.

Probably the most interesting genus resolved here as nonmonophyletic is Netrium—species analyzed form three independent branches, each characterized by a different number of chloroplasts per cell (1, 2, or 4), differing positions of the nucleus in the cell, and varying nuclear behavior during cytokinesis ( Pickett-Heaps 1975 Jarman and Pickett-Heaps 1990 unpublished observations). The three Netrium branches occupy a key position between the other Zygnematales and the Desmidiales, supporting previous rooted analyses containing only one Netrium species (as sister of the Desmidiales [ McCourt et al. 2000 Gontcharov, Marin, and Melkonian 2003]). In the latter publication, we erroneously reported two species of Netrium as having identical SSU rDNA sequences however, these sequences actually originated from the same culture, Netrium interruptum strain M 1021. Our expanded taxon sampling in this study reveals the Desmidiales as originating from a paraphyletic stock of derived unicellular Zygnematales (i.e., Netrium and Roya branches).

The Desmidiales, a clade defined by derived cell-wall characters, is well supported (but not in the rbcL phylogeny [see above]). The molecular phylogeny within the Desmidiales as revealed here and by previous studies ( McCourt et al. 2000 Gontcharov, Marin, and Melkonian 2003) reflects the increasing complexity of the cell-wall ultrastructure. Among the four families described, the Gonatozygaceae and Closteriaceae are confirmed, whereas the concept of the Peniaceae (cell wall consisting of several segments separated by shallow groove(s) simple pores perforating only the outer cell wall layer) and the Desmidiaceae (constricted cells composed of two semicells with complex [simple in Phymatodocis < Engels and Lorch 1981>] cell wall pores) are in need of revision. Two of three species of Penium analyzed form a robust clade with the Desmidiaceae (DESM). The third Penium species (P. spirostriolatum) forms a clade with DESM, although no morphological synapomorphy is presently known. Because of its simple cell-wall structure, Penium is usually not regarded as closely related to the Desmidiaceae.

Bayesian Phylogenetics

Bayesian inference, a recently introduced method for inferring molecular phylogenies ( Huelsenbeck and Ronquist 2001 Rannala and Yang 1996), provides a statistical confidence measure (PP) for branches and is much faster than ML bootstrap analysis. However, PP values are often much higher than ML BP and thus, the reliability of this method has recently been controversely discussed ( Huelsenbeck et al. 2002 Suzuki, Glazko, and Nei 2002 Alfaro, Zoller, and Lutzoni 2003 Douady et al. 2003). Based on simulation studies or using real sequence data, some authors considered ML BP as too conservative ( Hillis and Bull 1993 Murphy et al. 2001 Wilcox et al. 2002 Alfaro, Zoller, and Lutzoni 2003), whereas others concluded that PP is too optimistic ( Suzuki, Glazko, and Nei 2002). In our empirical study, the level of support for branches by PP or BP is roughly similar, although we also found several branches with significant PP support (≥0.95), which were not substantiated by significant BP values. One branch in the rbcL phylogeny, namely the branch separating CL and Roya/GON ( fig. 2), defines an artificial divergence that does not exist in the SSU rDNA topology and in the combined analysis (see Results). This artificial branch receives no BP values in the rbcL analysis, but considerable support by Bayesian inference (PP = 0.99 [ fig. 2]). It is known that Bayesian analysis is sensitive to small-model misspecifications ( Waddell, Kishino, and Ota 2001 Buckley 2002, Buckley et al. 2002), here probably related to the individual long-branch taxa Mesotaenium endlicherianum and Netrium oblongum SVCK255 and the high level of homoplasy in the rbcL gene. We conclude that Bayesian inference can be positively misleading as exemplified in our case study and suggest that PP support should always be confirmed by traditional bootstrap analyses.

Present address: Institute of Biology and Soil Science, Vladivostok, Russia.


Watch the video: Phylogenetic Insights into the Endophyte Symbiosis using PacBio Ribosomal DNA Sequencing (December 2022).