ORIGINAL ARTICLE

Molecular phylogeny, population genetics, and evolutionof heterocystous cyanobacteria using nifH gene sequences

Prashant Singh & Satya Shila Singh & Josef Elster &

Arun Kumar Mishra

Received: 10 May 2012 /Accepted: 26 September 2012 /Published online: 23 October 2012# Springer-Verlag Wien 2012

Abstract In order to assess phylogeny, population genetics,and approximation of future course of cyanobacterial evolu-tion based on nifH gene sequences, 41 heterocystous cyano-bacterial strains collected from all over India have been usedin the present study.NifH gene sequence analysis data confirmthat the heterocystous cyanobacteria are monophyletic whilethe stigonematales show polyphyletic origin with grave inter-mixing. Further, analysis of nifH gene sequence data usingintricate mathematical extrapolations revealed that the nucle-otide diversity and recombination frequency is much greaterin Nostocales than the Stigonematales. Similarly, DNA diver-gence studies showed significant values of divergence withgreater gene conversion tracts in the unbranched (Nostocales)than the branched (Stigonematales) strains. Our data stronglysupport the origin of true branching cyanobacterial strainsfrom the unbranched strains.

Keywords Evolution . Heterocystous cyanobacteria . nifHgene .Nucleotide diversity . Phylogeny . Population genetics

Introduction

Cyanobacteria are oxygenic, photosynthetic prokaryotesthat can be found ubiquitously in almost every realm ofthe earth (Henson et al. 2004). One of the surprising featuresrelated to the evolution of cyanobacteria is the similarity ofthe fossilized forms to the present day cyanobacterial spe-cies (Henson et al. 2002), thus indicating a sluggish pace ofevolutionary advancement.

Traditionally, cyanobacteria have been classified as algaeand were consequently grouped with plants due to its pho-tosynthetic ability, its possession of a distinct cell wall andthe presence of chlorophyll a as the main light-harvestingmolecule. Hence, early classificatory schemes of cyanobac-terial taxonomy were constructed with a botanical ratherthan bacterial criterion (Henson et al. 2002). This groupingof cyanobacteria with the plants was considered anomalousby microbiologists all over the world. A major drawback ofthis scheme was that it did not represent the true evolution-ary relationships and phylogeny within the cyanobacteriallineage, hence then arose the need for a bacteriologicalscheme of classification. This classificatory design wasbased primarily on the morphological attributes and thedevelopmental characteristics of the cyanobacterial genera(Rippka et al. 1979; Rippka 1988; Rippka and Herdman1992; Castenholz 2001). According to this format, five sec-tions of cyanobacteria were formally recognized. Section Icomprised of unicellular cyanobacteria that divided by binaryfission or budding. Section II consisted of unicellular formsthat divided bymultiple fission, resulting into the formation ofbaeocytes. From Section III onwards, the unicellularity of thecyanobacteria evolved into the formation of the filamentousforms. Members of Section III were filamentous but non-

Handling Editor: Peter Nick

P. Singh :A. K. Mishra (*)Laboratory of Microbial Genetics, Department of Botany,Banaras Hindu University,Varanasi 221005, Indiae-mail: akmishraau@rediffmail.com

A. K. Mishrae-mail: akmishraau@hotmail.com

S. S. SinghDepartment of Botany, Guru Ghasidas Vishwavidyalaya,Bilaspur, Chhattisgarh, India

J. ElsterCentre for Polar Ecology, Faculty of Science,University of South Bohemia,České Budĕjovice, Czech Republic

J. ElsterCentre for Phycology, Institute of Botany,Academy of Sciences CR,Třeboň, Czech Republic

Protoplasma (2013) 250:751–764DOI 10.1007/s00709-012-0460-0

heterocystous cyanobacteria that proliferated by trichomebreakage. Members of Sections IV and V were constituted bythe heterocystous cyanobacteria that were characterized bytheir ability to develop heterocysts and akinetes. They werepurely filamentous forms in which hormogonia formation wasseen as the prime mode of reproduction. Now, on the basis ofthe plane of division in the heterocystous clan, the two sectionswere further subdivided into two sub-sections, viz., Nostocalesand the Stigonematales. Section IV, i.e., the nostocalean mem-bers, comprised of strains that divided in only one plane, whileSection V, i.e., the stigonematalean line, constituted of strainsthat had the ability to divide in more than one plane (Rippka etal. 1979; Rippka 1988; Rippka and Herdman 1992; Castenholz2001).

A major drawback though of the morphological and thephysiological plan of taxonomy that has come into existenceis the plasticity of these attributes with environmental andculture variations (Rippka et al. 1979; Mollenhauer 1988;Rippka 1988). It has been vehemently argued that the cya-nobacterial classifications based on morphological charac-teristics may not show the true and accurate phylogeneticrelationships within the lineage because some of the mor-phological characters traditionally used to delineate generaand species are phenotypically plastic and can vary withenvironmental conditions (Henson et al. 2002). This notionhas been raised by a number of workers all across the globeand a lot of work has been done to prove the inaccuracy andinadequateness of the morphological scheme of classifica-tion (Rippka et al. 1979; Mollenhauer 1988; Rippka 1988;Komárek and Anagnostidis 1989; Stuken et al. 2006). It hasbeen reported that as many as 50 % of the cyanobacterialstrains found in culture collections all over the world havebeen misidentified and wrongly marked, thus leading tohighly erroneous phylogenic and diversity reports(Komárek and Anagnostidis 1989).

Nitrogen fixation (or diazotrophy) is the phenomenon of theutilization of atmospheric nitrogen (N2) to reduced forms likeammonia (NH3) (Postgate 1982). Chemically, in nature, a smallamount of atmospheric dinitrogen is reduced by lightning;however, the majority is reduced by prokaryotes (Postgate1982). The enzyme complex nitrogenase is responsible fornitrogen fixation. Nitrogenase is a complex consisting of twomajor subunits: dinitrogenase and dinitrogen reductase. Thesesubunits of nitrogenase are encoded by the nifHDK operon.Dinitrogenase is a tetramer with a molybdenum–iron (MoFe)cofactor. It is composed of two identical subunits encoded bythe nifD gene (Lammers and Haselkorn 1984) and the othertwo identical subunits are encoded by the nifK gene.Dinitrogenase binds atmospheric dinitrogen (N2) and is respon-sible for the transfer of electrons to it (Postgate 1982).Dinitrogenase reductase contains an iron cofactor and is com-posed of two identical subunits encoded by the nifH gene(Mevarech et al. 1980). It contains an iron–sulfur (4F–4S)

cofactor, which binds the subunits of nitrogenase, and is re-sponsible for mediating the ATP-dependent transfer of elec-trons to the dinitrogenase tetramer.

It is also essential to analyze the molecular data in syncwith bioinformatics and biostatistics. This facilitates theproper assessment of the DNA sequences along with high-lighting the environmental and geographical variations.Biostatistical tools developed for population genetics canbe used for deciphering the phylogenetic information inorder to assess the community structure (Yannarell et al.2006). Some of the major breakthroughs that this sort ofanalyses can provide is regarding crucial and very close toaccuracy information about the composition of the cyano-bacteria in the system under observation, the role andinsights about the geographic and environmental factors,and finally the relatedness of the ecological changes withthe molecular chronometers. Thus, extrapolation of DNAsequence data into mathematical form using proper pro-grams could help us in evaluating the evolutionary pace ineven selective group of organisms like heterocystous cya-nobacteria which vary widely in habitat along with beingrepresentatives of very diverse ecological changes.Community analyses have been deciphered using this ap-proach (Yannarell et al. 2006), but this has also been foundto be effective in related organisms isolated from wide-ranging habitats.

In this study, we aim to study the phylogeny and molec-ular diversity of heterocystous cyanobacteria using the nifHgene. An overall assessment of the diversity has beenattempted using a concerted approach based on morphology,molecular biology, bioinformatics and population geneticstools. Forty-one heterocystous cyanobacterial strains isolat-ed from all over India have been used in this study to assessthe phylogenetic relatedness and molecular diversity of 12genera of cyanobacteria along with the bioinformatics andgenetics studies. The objective of this work was to assess thephylogeny and the diversity of the heterocystous cyanobac-teria based on the nifH gene sequences. We also tried toinvestigate the nucleotide diversity amongst the heterocys-tous cyanobacteria based on the nifH gene sequence datathat we generated. Thus, we investigated both the molecularphylogeny and diversity of our isolates using a polymerasechain reaction (PCR)-based approach along with the supportof biostatistics and bioinformatics.

Materials and methods

Growth and maintenance of cyanobacteria

Forty-one heterocystous cyanobacterial strains collected fromdifferent geographical areas of India (Table 1; Fig. 1) weregrown axenically in 100 ml basal medium (BG-110 medium)

752 P. Singh et al.

(Rippka et al. 1979) in Erlenmeyer flasks (capacity 250 ml).Cyanobacterial strains were identified by the keys ofDesikachary (1959).

The pH of the medium was adjusted to 7.2 and thecultures were maintained in a culture room under illu-mination of approximately 50–55 μEm−2 s−1 with aphotoperiod of 14/10-h light/dark cycle at 28±2 °C.Subculturing of the strains to fresh liquid cultures wasaseptically performed under a laminar flow so as tomaintain uniform inoculums. Cultures were shaken twicea day. Microphotography of the pure axenic cultureswas done using Advanced Olympus Cat-cam fittedalong with the microscope (Fig. 2).

Genomic DNA isolation and PCR conditions

The DNA was isolated from only 8–10-day-old culturesusing Himedia Ultrasensitive Spin Purification Kit (MB505).The DNA eluted was stored at −20 °C. Degenerateprimers flanking a conserved region of the nifH genewere selected and cross-checked using Blastp algorithmin the NCBI portal. The primers used were nifHf (5′-CGTAGGTTGCGACCCTAAGGCTGA-3′) and nifHr(5′-GCATACATCGCCATCATTTCACC-3′), and theytargeted a fragment of approximately 297 bp (Guggeret al. 2005). The PCR amplification of nifH was

performed in 25-μl aliquots containing 10–20 ng DNAtemplate, 0.4 μM of each primer, 1.5 mM MgCl2,200 μM dNTPs, and 1 U/μl Taq polymerase. The tem-plate was initially denatured at 95 °C for 5 min. Thiswas followed by 30 cycles of denaturation for 15 s at95 °C, 30 s of annealing at 48 °C (except in the case ofNostoc calcicola, Scytonema bohnerii, and Nostochopsissp. where finally a gradient PCR resulted into anannealing temperature of 51.6 °C) and 1 min of exten-sion at 72 °C, followed at last by the final extensionstep of 5 min at 72 °C. The amplified products werevisualized on Bio Rad Gel Documentation system afterrunning in 1.2 % agarose gels.

Sequence analyses and construction of phylogenetic trees

The sequences obtained were then subjected to Blastn(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and submitted tothe NCBI database using the submission tool Sequin.Before submission, the sequences were annotated for thecoding regions using the NCBI ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf) and the ExpasyProteomics Server (http://expasy.org). The coding regionswere selected to the best of the suitability without anymanual editing of the original sequences. The sequenceswere then used to construct the phylogenetic tree using

Table 1 Cyanobacterial strains isolated from different habitats all over India along with NCBI accession numbers

Strain no. Cyanobacteria Accession number Strain no. Cyanobacteria Accession number

1 Anabaena doliolum Ind1 JQ627820 22 Scytonema bohnerii Ind24 JQ627811

2 Anabaena doliolum Ind2 JQ627819 23 Scytonema bohnerii Ind25 JQ246563

3 Anabaena oryzae Ind3 JQ627818 24 Fischerella sp. Ind26 JQ246559

4 Anabaena oryzae Ind4 JQ627817 25 Fischerella sp. Ind81 JQ246560

5 Anabaena sp. Ind5 JQ246561 26 Nostochopsis sp. Ind28 JQ246566

6 Anabaena sp. Ind6 JQ246562 27 Mastigocladus laminosus Ind29 JQ246568

7 Anabaenopsis sp. Ind8 JQ246569 28 Nostoc calcicola Ind30 JQ246547

8 Calothrix brevissima Ind9 JQ246550 29 Nostoc calcicola Ind31 JQ627821

9 Calothrix brevissima Ind10 JQ627816 30 Nostoc calcicola Ind32 JQ627822

10 Calothrix sp. Ind11 JQ246551 31 Nostoc muscorum Ind33 JQ246553

11 Cylindrospermum muscicola Ind12 JN652137 32 Nostoc muscorum Ind34 JQ627823

12 Cylindrospermum muscicola Ind13 JQ627815 33 Nostoc sp. Ind36 JQ246554

13 Cylindrospermum sp. Ind14 JQ627814 34 Nostoc sp. Ind37 JQ246556

14 Cylindrospermum stagnale Ind15 JQ246567 35 Nostoc sp. Ind39 JQ246555

15 Tolypothrix tenuis Ind16 JQ246570 36 Nostoc sp. Ind40 JQ246557

16 Tolypothrix nodosa Ind17 JQ246558 37 Nostoc sp. Ind40.1 JQ246548

17 Westiellopsis sp. Ind19 JQ246549 38 Nostoc sp. Ind40.2 JQ246546

18 Westiellopsis sp. Ind20 JQ627813 39 Nostoc spongiaeforme Ind41 JQ627824

19 Hapalosiphon welwitschii Ind21 JQ627812 40 Nostoc spongiaeforme Ind42 JQ246552

20 Hapalosiphon welwitschii Ind22 JQ246565 41 Anabaena sp. PCC 7120 Ind43 JQ627825

21 Hapalosiphon sp. Ind23 JQ246564 (Nostoc sp. PCC 7120 Ind43)

Phylogeny, population genetics and evolution of cyanobacteria 753

MEGA 5 (Tamura et al. 2011) in accordance with theinterior test of phylogeny using neighbor-joining algorithmand the Jukes–Cantor scheme of phylogeny.

Nucleotide diversity analyses

In order to obtain information about the population structure,the nucleotide diversity and the future course of evolution ofthe cyanobacterial lineage based on nifH gene sequences,statistical analyses were performed using the software

DnaSP 5.10 (Librado and Rozas 2009). A three-stage analysisfocusing individually on the unbranched (Nostocales), thebranched (Stigonematales), and finally the two in combinationwas done. Main parameters studied were the measurementof the molecular indices using the DNA sequence data(Table 2).

We calculated the nucleotide diversity (π) on the basis ofthe formula:

p ¼ k m=

Fig. 1 Map of heterocystous cyanobacteria collection sites (https://maps.g o o g l e . c om /m a p s / m s ? t =m&v p s r c = 6&m s a = 0&m s i d =209861985046989110332.0004b7584ca2b75492767&ie=UTF8&q=

Pallikaranai+Marsh+Reserve+Forest&cid=11522957690802974996&ll=23.885838,83.320313&spn=42.7894,86.572266&z=4)

754 P. Singh et al.

where k is the mean number of nucleotide differences, whilemis the total number of nucleotide positions. The variable k canbe defined by the formula given below:

k ¼ 2

n n� 1ð ÞXi<j

dij

where n is the number of sequences (i.e., the sample size) anddij is the number of nucleotide differences between sequencesi and j. The mean number of nucleotide differences is alsocomputable by the formula:

k ¼Xmi¼1

hi

Fig. 2 Microphotographs ofheterocystous cyanobacteria. 1A. doliolum Ind1, 2 A. doliolumInd2, 3 A. oryzae Ind3, 4 A.oryzae Ind4, 5 Anabaena sp.Ind5, 6 Anabaena sp. Ind6, 7Anabaenopsis sp. Ind8, 8 C.brevissima Ind9, 9 C.brevissima Ind10, 10 Calothrixsp. Ind11, 11 Cylindrospermummuscicola Ind12, 12 C.muscicola Ind13, 13Cylindrospermum sp. Ind14, 14Cylindrospermum stagnaleInd15, 15 Tolypothrix tenuisInd16, 16 Tolypothrix nodosaInd17, 17 Westiellopsis sp.Ind19, 18 Westiellopsis sp.Ind20, 19 Hapalosiphonwelwitschii Ind21, 20 H.welwitschii Ind22, 21Hapalosiphon sp. Ind23, 22 S.bohnerii Ind24, 23 S. bohneriiInd25, 24 Fischerella sp. Ind26,25 Fischerella sp. Ind81, 26Nostochopsis sp. Ind28, 27 M.laminosus Ind29, 28 N.calcicola Ind30, 29 N. calcicolaInd31, 30 N. calcicola Ind32,31 Nostoc muscorum Ind33,32 N. muscorum Ind34, 33Nostoc sp. Ind36, 34 Nostoc sp.Ind37, 35 Nostoc sp. Ind39, 36Nostoc sp. Ind40, 37 Nostoc sp.Ind40.1, 38 Nostoc sp. Ind40.2,39 Nostoc spongiaeformeInd41, 40 N. spongiaeformeInd42, 41 Anabaena sp. PCC7120 Ind43 (Nostoc sp. PCC7120 Ind43)

Phylogeny, population genetics and evolution of cyanobacteria 755

where hi is the heterozygosity at site i which is esti-mated as:

hi ¼ n

n� 11�

X4j¼1

x2ij

!In the preceding formula given, xij is the relative frequency ofnucleotide variant j (j01, 2, 3, and 4 corresponding to A, C, G,and T) at site i.

The complete analysis involved the DNA polymor-phism evaluation, searching the parsimony informativesites, calculating the nucleotide diversity per site (Pi),

Fig. 2 (continued)

756 P. Singh et al.

measurements of the theta per site from the total num-ber of mutations (Eta), estimating the number of nucle-otide differences (k), deciphering the recombinationprobabilities and finally studying the DNA divergencebetween the two populations selected (unbranched andbranched cyanobacteria).

Results

PCR of nifH gene fragments

PCR amplification of the nifH gene of the 41 heterocystouscyanobacteria was done with some strains needing gradientPCR standardizations. It was always found suitable in allcases to attempt a hot start PCR with the templates beingdenatured initially by using a heated lid in the PCR. It was

noticed that 0.4 μM primer concentration was always foundsuitable for PCR optimization.

Annotation of the sequences

The sequences obtained were annotated using the NCBIORF Finder and cross-checked using the Expasy proteomicsServer. Thereafter, the SQN files were prepared on the basisof the original sequences without any editing of the sequen-ces. The sequences were at last submitted to the NCBIdatabase and accession numbers were obtained.

Construction of the phylogenetic tree

The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei 1987). The optimal tree withthe sum of branch length 01.26186410 was drawn. The

Fig. 2 (continued)

Phylogeny, population genetics and evolution of cyanobacteria 757

confidence probability (multiplied by 100) where the interi-or branch length is greater than 0, as estimated using thebootstrap test (1,000 replicates), is shown next to thebranches [Dopazo 1994; Rzhetsky and Nei 1992]. The

evolutionary distances were computed using the Jukes–Cantor method (Jukes and Cantor 1969) and are in the unitsof the number of base substitutions per site. The analysisinvolved 46 nucleotide sequences. Codon positions included

Fig. 2 (continued)

758 P. Singh et al.

were first+second+third. All positions containing gaps andmissing data were eliminated. There were a total of 171positions in the final dataset. Evolutionary analyses wereconducted in MEGA5 (Tamura et al. 2011).

The tree obtained showed ample proof that there is aserious need for some classificatory reforms in the heterocys-tous cyanobacteria. Within the order Nostocales, there was a

broad intermixing of the genera as seen in the case of group D(Fig. 3). Group A comprised exclusively of members of theorder Stigonematales with representatives of the generaHapalosiphon, Nostochopsis, and Fischerella. Group B con-sisted exclusively of the genera Cylindrospermum, whilegroup C made an interesting cluster with Mastigocladus,Nostoc, and Fischerella. Group D comprised of Anabaena

Fig. 2 (continued)

Phylogeny, population genetics and evolution of cyanobacteria 759

sp. along with the genera Tolypothrix. Group E comprised ofAnabaena sp., Anabaena oryzae, and Anabaena doliolum.Group F consisted of a very perfect and tight clustering ofmembers of the order Nostoc. Group G consisted of N. calci-cola exclusively. Interestingly, Anabaenopsis sp. and Nostocsp. Ind 40 formed single clusters H and I, respectively. Ourown S. bohnerii isolates paired with the Scytonema sp. ofgenebank forming the cluster J. Intermixing was again ob-served very prominently in cluster K, where our Nostoc sp.Ind40.1 isolate paired with our Westiellopsis sp. 19 andWestiellopsis sp. 20 isolates. Finally, our Calothrix brevissimaisolates, Calothrix sp., and Calothrix sp. (MCC-3A) of thegenebank formed the last group L. Oscillatoria sp. PCC 6506was taken as an outgroup.

Population genetics analyses

In order to obtain information about the population struc-ture, the nucleotide diversity, and the future course of evo-lution of the cyanobacterial lineage based on nifH genesequences, statistical analyses were performed using thesoftware DnaSP 5.10 (Librado and Rozas 2009) (Table 2).The mathematical extrapolations showed unambiguous evo-lutionary patterns between two highly close and much de-bated groups of cyanobacteria, thus demonstrating that theNostocales or the simpler forms hold the key for decidingthe future course of evolution.

Discussion

Molecular phylogeny of heterocystous cyanobacteriausing the nifH gene

The cyanobacterial nifH gene has been well studied and wellcharacterized particularly in the members of the generaNostoc and Anabaena (Tamas et al. 2000). Gugger et al.(2002) studied the nifH gene of Cylindrospermopsis strainsisolated from four continents. In our case, we made anattempt to characterize heterocystous cyanobacteria thatrepresented 12 genera. The tree obtained, showed ampleevidence that there is a serious need for some classificatoryreforms in the heterocystous cyanobacteria. In our cluster A,we found the presence of the true branching cyanobacterianot very exclusively well separated from group B compris-ing of Cylindrospermum strains. The low bootstrap value of52 indicates the very loose and labile separation of the twogroups from each other. In group C, a mixed phylogeny wasseen with our own Fischerella isolate pairing with theuncultured Fischerella of genebank with a high bootstrapof 99. In this group only, our own Nostoc sp. was indiversification with the true-branching Mastigocladus lam-inosus. Group D was interesting as Anabaena Ind6 pairedwith Tolypothrix nodosa and Tolypothrix tenuis. This pair-ing of the unbranched cyanobacteria with the false-branchedstrains possibly suggests the evolutionary relatedness of

Table 2 Population geneticsanalysis of heterocystouscyanobacteria

Parameters Unbranched(members ofNostocales)

Branched(members ofStigonematales)

All strains

Polymorphic sites (segregating sites, S) 206 254 206

Parsimony informative sites 206 224 206

Nucleotide diversity per site (Pi) 0.62907 0.59486 0.65531

Theta per site from total (number of mutations, Eta) 0.68368 0.75965 0.67266

Average number of nucleotide (differences, k) 129.588 151.689 134.994

Recombinational analyses

Variance of the sample (distribution of kij, Sk2) 2,591.959 5,812.53 1,948.141

Theta per gene 129.588 5,812.53 1,948.141

R per gene 45.3 25.9 81.4

Estimate of R between adjacent sites 0.1673 0.0858 0.2916

Average nucleotide distance between the most distant sites 270.77 301.8 279.17

DNA divergence between the groups

Average number of nucleotide differences between groups 144.166

Number of net nucleotide subpopulations per sitebetween the groups, Da

0.08503

Number of gene conversion tracts identifiedbetween groups

63

Mutations polymorphic in group 1, butmonomorphic in group 2

155

Mutations polymorphic in group 2, butmonomorphic in group 1

28

760 P. Singh et al.

false-branching cyanobacteria with the unbranched ones.This group also indicates the earlier suggested polyphyleticorigin of Anabaena (Neilan et al. 1995). Groups E and Fshow very perfect Anabaena and Nostoc clusters, thus indi-cating clear-cut sequence-based differences amongst the twogenera. nifD gene studies (Henson et al. 2002) had alsosuggested the distinctiveness of both genera, and our studyalso supports it, at least when considering the nifH gene.Earlier nifH gene studies (Tamas et al. 2000; Zehr et al.1997) had failed to distinguish the strains, but here in ourstudy we do support the separation of Nostoc and Anabaena

as mentioned in the earlier classificatory schemes (Castenholz2001). Groups G, H, and I show very small clusters of N.calcicola, Anabaenopsis, and Nostoc. These groups owe theirindividuality probably to the environmental and geographicvariations from the rest of their supposedly nearer strains.Groups G, H and I truly reflect that why there have beenvehement claims for a classificatory reconsideration (Rippka1988). Culture conditions, geographic contours, and envi-ronmental pressures are all plausible reasons that taketheir toll on deciding the phenotype of an organism, butthis phenotypic plasticity is very much variable and this

Fig. 3 Phylogenetic tree showing the clustering of heterocystous cyanobacteria based on the nifH gene analysis

Phylogeny, population genetics and evolution of cyanobacteria 761

explains the reasons for groups G, H, and I. Group Jcomprises the false-branching strains of Scytonema, andthis group also supports the need for more molecularstudies on cyanobacterial systematics. We have alreadyseen Tolypothrix isolates pairing with Anabaena in groupD. This mispriming of members of supposedly the samefamilies again finds its origin in them being from differentgeographical origins. Group K once again showed a veryclear-cut intermixing of true-branching cyanobacteria withunbranched cyanobacteria. Our Westiellopsis isolates clus-tered with the Nostoc isolate, and very interestingly eventhe bootstrap value was robust enough at 99.

The final group L comprises of isolates of Calothrixpairing with one another. In context of all the cited findings,we seem sure that there is a need for a rethinking of theclassificatory schemes. The orders Nostocales andStigonematales have been found to be intermixed with oneanother (Gugger and Hoffmann 2004; Henson et al. 2004).At places where they seem to have appeared close amongstthemselves (as in group A) the low bootstrap values make itdifficult to make these findings very strong and supportive.The grouping of Nostoc sp. PCC 7120 strains with oneanother supports the assignment of this strain to the generaNostoc rather than Anabaena. Although the positioning ofthis strain has been long debated (Tamas et al. 2000) but ournifH based analyses supports the assignment of strain PCC7120 to Nostoc rather than Anabaena (Henson et al. 2002).The tight clustering of Nostoc strains with one anotherfurther favors the clear cut division Nostoc and Anabaenainto separate genera (Henson et al. 2002). A very interestingfeature is the tight clustering of Fischerella sp. with mem-bers of order Stigonematales. This supports some earlierreports (Zehr et al. 1997) which support the inclusion ofthis species into the true branching forms. In the group Calso which has shown intermixing of Nostocales andStigonematales, the placing of Nostoc sp. Ind40 can beregarded as a result of the environmental pressures andgeographic separation. We could not distinguish very clearlyin between the true branching forms (Zehr et al. 1997) butstill the small pattern obtained does support the establish-ment of Fischerella as a true branching cyanobacterium.There have been reports of the heterocystous clade beingmonophyletic (Rajaniemi et al. 2005) but intermixed(Gugger and Hoffmann 2004; Henson et al. 2004) and wefound this hypothesis to be correct. The strain Anabaenopsisclustered into a single and individual group thus, establish-ing once gain its difference even in terms of the geneticarchitecture as reported earlier (Iteman et al. 2002) by 16SrRNA and RFLP analyses. The group L that containedexclusively the Calothrix strains exhibited a very tight clus-tering. This finding using the nifH gene seems to be in syncwith earlier reports (Mishra et al. 2012; Sihvonen et al.2007) where separate clustering of this genus was observed.

The taxonomic position of this cyanobacterium seems to becorrect and it definitely holds both phenotypic and genotyp-ic incoherence with other members of the order Nostocalesand thus can be easily grouped into a separate genera. Thepositioning of Westiellopsis sp. in our study seemed to be insupport of the polyphyly of the subsection V of the tradi-tional scheme of cyanobacterial taxonomy. Earlier reportsusing the nifD gene (Henson et al. 2004) and the nifH gene(Zehr et al. 1997) have also suggested towards theStigonematales being polyphyletic. Although our own 16SrRNA reports (Mishra et al. 2012) have suggested towardsthe monophyly of the subsection V, this nifH gene analysesdoes not seem to be in congruence with this report though.

Our nifH analysis supports the polyphyly of theStigonematales and we report a clear inter-mixing betweenthe members of the subsections IVand V. The genera Nostocseems to have been the most genetically diverse in terms ofthe nifH gene and was found to be clustering with membersof false-branching strains and even with the true-branchinggenera.

Population genetics analyses

Our phylogenetic analyses using the nifH gene sequenceshave shown the heterocystous clade to be monophyletic, i.e.,origin from one common ancestor. We tested our phyloge-netic findings based on simple mathematical tools and foundour phylogenetic findings to be correct. The analyses of thesampled taxa as two major groups, viz., Nostocales andStigonematales, on the basis of the nucleotide sequencesresulted into some very interesting findings. The nucleotidediversity per site (Pi) was found to be greater in theNostocales order in comparison to the Stigonematales.This directly points out towards the role of this order inconstituting the skeleton of the course of evolution of het-erocystous cyanobacteria. Our analyses also pointed outtowards the more number of average nucleotide differences(k) in the Stigonematales in comparison to the Nostocales.This may be a consequence of the singleton variable sitesthat were 30 in the case of the true-branching cyanobacteriaand none in case of the unbranched cyanobacteria. To testour calculations, we conducted the recombination analysesin between the groups and estimated the value of R per gene tobe greater in the Nostocales order in comparison to theStigonematales. We also calculated R between adjacent sitesand found this also to be greater in the unbranched filamentousand lesser in the branched cyanobacteria. On calculating theaverage nucleotide distance between the most distant sites, theNostocales order clearly pointed out towards its greater evolu-tionary tendency in comparison to the order Stigonematales.Our recombination analyses clearly shows that the orderNostocales could be controlling the future course of evolutionof heterocystous cyanobacteria and hence constitutes the main

762 P. Singh et al.

root from which the other advanced cyanobacteria eitherwould have evolved in the past or may evolve in the future.The role of the non-heterocystous cyanobacteria remains to betested, but certainly we have proven mathematically that thelower forms have greater and faster evolutionary tendenciesthan the higher and the advanced forms. On studying the DNAdivergence between the groups, the hypothesis of Nostocalesbeing more progressive gained more momentum. Out of thetotal number of 63 gene conversion tracts identified, we found55 of them to be in the order Nostocales and only eight werefound in the Stigonematales. In our case, gene conversiontracts can be regarded as simply those segments of the se-quence/gene that may participate in framing the next course orstep of evolution of new patterns or forms. They can also beregarded as the hot-spots of evolution. Our studymay have onelacuna in including fewer members of the true-branchingforms, but the recombination analyses and DNA divergencestudies proved that the unbranched filamentous forms do havegreater momentum of evolution. We also identified the muta-tions polymorphic in Nostocales and monomorphic inStigonematales and found these to be 155. On the oppositeside, i.e., mutations polymorphic in Stigonematales andmonomorphic in Nostocales, the value was found to be just28. Thus, on an overall scale, we have gained immense datathat support the enhanced role of the order Nostocales incomparison to the Stigonematales. Our nifH gene analysisdefinitely recognizes the importance of the unbranched cya-nobacteria in deciding the course of evolution and origin ofnew species of cyanobacteria.

After analyses of the phylogenetic and the nucleotidediversity of the selected heterocystous cyanobacteria onthe basis of the nifH gene characterization, we do stronglysupport the monophyly of the heterocystous cyanobacteriawith intermixing. The order Stigonematales was found to bepolyphyletic in our study, thus deviating from both our ownand of other workers’ structural gene analyses. Our resultscorroborate fully with the reports of other groups workingon the cyanobacterial nifH genes. The population geneticsanalysis has given strong evidence towards the evolutionarytendencies of the order Nostocales. Mathematical extrapo-lations of the nifH sequence data have vehemently sug-gested the rise of the branched forms from the unbranchedones. The possible course of future evolution would defi-nitely imprint the unbranched, simpler cyanobacteria at theroots from which the advanced, multicellular, and branchedforms would originate.

Acknowledgments We are thankful to the Department of Scienceand Technology, Ministry of Science and Technology, for providingfinancial support in the form of a project. One of us (PS) is alsothankful to CSIR, New Delhi, for their financial support in the formof SRF. The Head, Department of Botany, Banaras Hindu University,Varanasi, India, is gratefully acknowledged for providing laboratoryfacilities.

Conflict of interest The authors declare that they have no conflict ofinterest.

References

Castenholz RW (2001) Phylum BX. Cyanobacteria, oxygenic photo-synthetic bacteria. In: Boone DR, Castenholz RW (eds) Bergey’smanual of systematic bacteriology, vol 1. Springer, New York, p721

Desikachary TV (1959) Cyanophyta, part I and II. Indian council ofagricultural research, New Delhi

Dopazo J (1994) Estimating errors and confidence intervals for branchlengths in phylogenetic trees by a bootstrap approach. J Mol Evol38:300–304

Gugger M, Hoffmann L (2004) Polyphyly of the true branching cya-nobacteria (Stigonematales). Int J Syst Evol Microbiol 54:349–357

Gugger M, Lyra C,Henrikson P, Coute A, Humbert JF, SihvonenK (2002)Phylogenetic comparison of the cyanobacterial genera Anabaena andAphanizomenon. Int J Syst Evol Microbiol 52:1867–1880

GuggerM,Molica R, Berre BL, Dufour P, Bernard C, Humbert JF (2005)Genetic diversity of Cylindrospermopsis Strains (Cyanobacteria)isolated from four continents. Appl Environ Microbiol 71:1097–1100

Henson BJ, Watson LE, Barnum SR (2002) Molecular differentiationof the heterocystous cyanobacteria, Nostoc and Anabaena, basedon complete NifD sequences. Curr Microbiol 45:161–164

Henson BJ, Hesselbrock SM, Watson LE, Barnum SR (2004)Molecular phylogeny of the heterocystous cyanobacteria (subsec-tions IV and V) based on nifD. Int J Syst Evol Microbiol 54:493–497

Iteman I, Rippka R, Tandeau de Marsac N, Herdman M (2002) rDNAanalyses of planktonic heterocystous cyanobacteria, including mem-bers of the genera Anabaenopsis and Cyanospira. Microbiol148:481–496

Jukes TH, Cantor CR (1969) Evolution of protein molecules. In:Munro HN (ed) Mammalian protein metabolism. Academic,New York, pp 21–132

Komárek J, Anagnostidis K (1989) Modern approach to the classifica-tion system of cyanophytes 4 Nostocales. Arch Hydrobiol Suppl82:247–345

Lammers P, Haselkorn R (1984) Sequence of the nifD gene coding for theα subunit from the cyanobacterium Anabaena. Cell 44:905–911

Librado P, Rozas J (2009) DnaSP v5: a software for comprehensiveanalysis of DNA polymorphism data. Bioinformatics 25:1451–1452

Mevarech M, Rice D, Haselkorn R (1980) Nucleotide sequence of acyanobacterial nifH gene coding for nitrogenase reductase. ProcNatl Acad Sci USA 77:6476–6480

Mishra AK, Shukla E, Singh SS (2012) Phylogenetic comparisonamong the heterocystous cyanobacteria based on a polyphasicapproach. Protoplasma. doi:10.1007/s00709-012-0375-9

Mollenhauer D (1988) Nostoc species in the field. Arch HydrobiolSuppl 80(315):326

Neilan BA, Jacobs D, Goodman AE (1995) Genetic diversity andphylogeny of toxic cyanobacteria determined by DNA polymor-phisms within the phycocyanin locus. Appl Environ Microbiol61:875–3883

Postgate JR (1982) The fundamentals of nitrogen fixation. CambridgeUniversity Press, Cambridge

Rajaniemi P, Hrouzek P, Kastovska K, Willame R, Rantala A, HoffmannL, Komárek J, Sivonen K (2005) Phylogenetic and morphologicalevaluation of the genera Anabaena, Aphanizomenon, Trichormus

Phylogeny, population genetics and evolution of cyanobacteria 763

and Nostoc (Nostocales, Cyanobacteria). Int J Syst Evol Microbiol55:11–26

Rippka R (1988) Recognition and identification of cyanobacteria.Methods Enzymol 167:28–67

Rippka R, Herdman M (1992) Pasteur culture collection of cyanobac-terial strains in axenic culture, catalogue and taxonomic hand-book. Institut Pasteur, Paris

Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979)Generic assignments, strain histories and properties of pure cul-tures of cyanobacteria. J Gen Microbiol 111:1–61

Rzhetsky A, Nei M (1992) A simple method for estimating and testingminimum evolution trees. Mol Biol Evol 9:945–967

Saitou N, Nei M (1987) The neighbour joining method—a new methodfor reconstructing phylogenetic tree. Mol Biol Evol 4:406–425

Sihvonen LM, Lyra C, Fewer DP, Rajaniemi-Wacklin P, Lehtimáki JM,Wahlsten M, Sivonen K (2007) Strains of the cyanobacterialgenera Calothrix and Rivularia isolated from the Baltic Sea dis-play cryptic diversity and are distantly related to Gloeotrichia andTolypothrix. FEMS Microbiol Ecol 61:74–84

Stuken A, Rucker J, Endrulat T, Preussel K, HemmM,Nixdorf B, KarstenU, Wiedner C (2006) Distribution of three alien cyanobacterialspecies (Nostocales) in northeast Germany Cylindrospermopsis raci-borskii, Anabaena bergii and Aphanizomenon aphanizomenoides.Phycologia 45:696–703

Tamas I, Svircev Z, Andersson SGE (2000) Determinative value of aportion of the nifH sequence for the genera Nostoc and Anabaena(Cyanobacteria). Curr Microbiol 41:97–200

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011)MEGA5: molecular evolutionary genetics analysis using maxi-mum likelihood, evolutionary distance and maximum parsimonymethods. Mol Biol Evol 28:2731–2739

Yannarell AC, Steppe TF, Paerl HW (2006) Genetic variance in thecomposition of two functional groups (diazotrophs and cyanobac-teria) from a hypersaline microbial mat. Appl Environ Microbiol72:1207–1217

Zehr JP, Mellon MT, Hiorns WD (1997) Phylogeny of cyanobacterialnifH genes: evolutionary implications and potential applicationsto natural assemblages. Microbiol 143:1443–1450

764 P. Singh et al.