Morphological and molecular characterization of

Phycologia (2007) Volume 46 (4), 000–000

Published XXX XXX 2007

Morphological and molecular characterization of selected desert soil cyanobacteria: three species new to science including Mojavia pulchra gen. et sp. nov. ˇ EHA´KOVA´1,2*, JEFFREY R. JOHANSEN3, DALE A. CASAMATTA4, LI XUESONG3 KLA´RA R

AND

JASON VINCENT3

ˇ eske´Budeˇjovice 37005, Czech Republic Institute of Hydrobiology, AS CR, Na sa´dka´ch 7, C 2 Institute of Botany, AS CR, Dukelska´ 145, Trˇebonˇ 37982, Czech Republic 3 Department of Biology, John Carroll University, University Heights, OH 44118, USA 4 Department of Biology, University of North Florida, Jacksonville, FL 32224-2660, USA

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ˇ EHA´KOVA´, J.R. JOHANSEN, D.A. CASAMATTA, L. XUESONG AND J. VINCENT. 2007. Morphological and molecular K. R characterization of selected desert soil cyanobacteria: three species new to science including Mojavia pulchra gen. et sp. nov. Phycologia 46: xxx–xxx. 10.2216/XX–XX.1

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Four Nostocacean species from desert soils of the western United States, including the phycobiont of the lichen Collema tenax, were studied. Our strains could be forced into morphospecies previously described from Europe, but phylogenetic analysis indicated that they belonged in separate, distinct, and previously undescribed taxa. Partial 16S rRNA sequences of the strains CM1-VF10, CM1-VF14, CNP-AK1 and JT2-VF2 were determined and aligned with published Nostoc sequences from GenBank and our lab, as well as other Nostocales. All aligned sequences were analysed using parsimony, distance, and maximum likelihood methods, and trees based on three separate data sets were generated. Full 16S-23S internal transcribed spacer (ITS) regions were also characterized for our strains, and secondary structures of the ITS region were compared among these and N. commune and N. punctiforme. Intragenomic variability was documented among ITS regions in different operons for these taxa. One of the four strains (JT2-VF2) is distinct from Nostoc by both morphological and molecular criteria and is described as Mojavia pulchra gen. et sp. nov. Two other strains (CM1-VF10 and CM1-VF14) are described as Nostoc indistinguendum sp. nov. and Nostoc desertorum sp. nov., respectively. According to both morphological and molecular characteristics, the phycobiont of C. tenax is not N. commune, N. sphaericum or N. punctiforme as variously suggested in the lichenological literature, and the older name for this taxon, Nostoc lichenoides, is consequently validated in this paper. KEY WORDS: Collema, Cyanobacteria, Clark Mountains, ITS secondary structure, Microbiotic soil crust, Mojavia, Nostoc, rRNA operons

INTRODUCTION

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Nostoc is a well-known, widespread genus within the cyanobacteria. It occurs in freshwater aquatic, hydroterrestrial, and euterrestrial habitats throughout the world (Geitler 1932). Geoffroy is credited with the first use of the name by Drouet (1978 – as Nostoc Geoffroy ex Linnaeus), but due to Article 13.1 of the International Code of Botanical Nomenclature (Greuter et al. 2000), valid publication for the heterocytous cyanobacteria begins with Bornet and Flahault (1886–1888). This ruling makes Nostoc Vaucher ex Bornet et Flahault the valid name for the genus, with Nostoc commune Vaucher (1803) ex Bornet et Flahault (1886–1888) serving as the generitype. The genus is problematic due to an absence of derived characteristics with which to define species, as well as an at times complicated life history that is seldom described in the classical literature. Of all the Nostocales, Nostoc is one of the most lacking in morphological synapomorphies. It is the genus which lacks true branching, false branching, special positioning of the heterocyte, specialized end cells, tapering of the trichomes, heteropolarity, and aerotopes. The only potentially apomorphic character is production of a mucilaginous investment, this latter trait being the * Corresponding author ([email protected]).

primary basis of separation of Trichormus from Nostoc. Species within the genus have been separated primarily on the basis of cell dimensions, macrocolony morphology, and ecology. There is considerable overlap in these characters among many of the morphospecies that have been described. The group is confusing enough for Drouet (1978) to have combined all previously described species into N. commune. Researchers studying the cyanobacteria of desert soil crusts have generally adopted the names used for terrestrial Nostoc species described from humid European soils. Commonly reported taxa include N. commune, N. punctiforme (Ku¨tz.) Hariot, N. muscorum Ag. ex Born. et Flah., N. paludosum Ku¨tz. ex Born. et Flah., and N. piscinale Ku¨tz. ex Born. et Flah. (Johansen et al. 1981, 1984, 1993; Flechtner et al. 1998). We recently isolated a number of Nostoc-like strains from desert soils. These strains have cell dimensions similar to species described from Europe, particularly N. commune and N. punctiforme, but inhabit markedly different biotopes. This paper reports on the morphology, life cycle, and sequence of ribosomal RNA genes (16S and 16S-23S internal transcribed spacer [ITS] regions) of four of these isolates. On the basis of our analysis, we describe as new species Nostoc desertorum sp. nov., Nostoc indistinguendum sp. nov., and Mojavia pulchra gen. et sp. nov. and validate Nostoc lichenoides Vauch. We 0

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Table 1. Sources for 16S rRNA sequences generated in the Johansen laboratory (John Carroll University) for this study. Accession numbers in parentheses represent 16S-23S ITS gene sequences. Under origin, accession numbers for the Herbarium of Nonvascular Cryptogams (BRY) are given for five strains deposited in that collection. Strain Nostoc lichenoides CNP-AK1 Nostoc cf. lichenoides JT1-VF3 Nostoc cf. lichenoides JT1-VF7 Nostoc indistinguendum CM1-VF10 Nostoc cf. indistinguendum FI5-VF12 Nostoc cf. indistinguendum FI5-VF1 Nostoc cf. indistinguendum FI5-VF4 Nostoc desertorum CM1-VF14 Nostoc commune EV1-KK1 Mojavia pulchra JT2-VF2

Accession number AY577535 AY577532 AY577533 AY577538 AY577539 AY577541 AY577540 AY577537 AY577536 AY577534

(AY579896 to AY579898)

(AY579899 to AY579902) (AY579904, AY579905) (AY579903)

will use these taxonomic epithets in the remainder of the paper.

MATERIALS AND METHODS Isolation of strains Soil samples from arid regions in California and Utah were collected by J.R. Johansen and student collaborators and used for the isolation of cyanobacterial strains discussed in this paper. Nostoc commune EV1-KK1 was isolated from ˇ eske´ Budeˇjovice, Czech Republic, and a cement basin in C its identity as that taxon was confirmed by Dr. Jirˇi Koma´rek (Academy of Science, Trˇebonˇ, Czech Republic). All samples were dilution plated on to agar-solidified Z-8 medium (Carmichael 1986), and individual cyanobacterial isolates were made from these plates onto agar slants of Z-8 or N-free medium (Z-8 without nitrates). Cyanobacterial strains were maintained on agar-solidified Z8 or N-free media at 7uC on a 16:8-h light:dark cycle under fluorescent light (200 mEs21 cm22). Morphological observation CM1-VF10 and CM1-VF14 from Clark Mountains, San Bernardino County, California; JT2-VF2 from Cadiz Valley, Joshua Tree National Park, California; and CNP-AK1 from Canyonlands National Park, Utah, were spread on N-free agar-solidified medium in Petri dishes in five repetitions. Morphology was examined using an Olympus BH-2 compound photomicroscope with Nomarski DIC optics and a Nikon stereomicroscope. Observations were made throughout the life cycle, first every 3 days up to 3 weeks, then every 5 days up to 5 weeks, every 14 days up to 4 months, by which time material was scenescent, and finally at 6 months. Characteristics assessed were colour and shape of macrocolonies and microcolonies, shape and size of vegetative cells, heterocytes, akinetes and hormogonia, positions of heterocytes and akinetes within trichomes, arrangement of filaments in the mucilage sheath, and colour and structure of the sheath.


Origin

(AY579894, AY579895)

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Canyonlands National Park, UT, USA. BRY C-37687. desert soil, Joshua Tree National Park, CA, USA. desert soil, Joshua Tree National Park, CA, USA. desert soil, Clark Mountains, CA, USA. BRY C-37689. Desert soil, Fort Irwin, CA, USA desert soil, Fort Irwin, CA, USA desert soil, Fort Irwin, CA, USA desert soil, Clark Mountains, CA, USA. BRY C-37691. ˇ eske´ Budeˇjovice, Czech Republic. BRY C-37695. soil, C desert soil, Joshua Tree National Park, CA, USA. BRY C-37693.

Statistical analysis One-way analysis of variance (ANOVA) and the Tukey HSD multiple comparison test using Statistica for Windows 4.0 (StatSoft Inc., Tulsa, OK) were employed to evaluate differences in length and width of vegetative cells, heterocytes, and akinetes. Generally, 400 measurements for each strain were used for vegetative cells and heterocytes and a subset of 100 measurements for akinete dimensions. Given the size of the data set, we chose a 5 0.01 for these six tests. Molecular methods Molecular protocols, including primer sequences and temperature profiles, were identical to those reported in detail in Flechtner et al. (2002) and Boyer et al. (2002). Briefly, DNA was extracted using Cullings’s (1992) modification of the Doyle and Doyle (1987) technique. A long PCR beginning at base pair (bp) 359 in the 16S rRNA gene and ending at bp 45 in the 23S rRNA gene was first amplified. This amplification product was used for three subsequent reamplifications that amplified the 16S rRNA gene and associated 16S-23S ITS region. Reamplification products were subsequently cloned prior to sequence determination (TOPO-TA cloning kits, Invitrogen, Carlsbad, CA), a step especially necessary in determining the sequence of the ITS region of the multiple ribosomal operons in the Nostocales (Iteman et al. 2000; Boyer et al. 2001). Cloned plasmids containing inserts were isolated according to the instructions provided in the QIAprep Mini-prep kitH (Qiagen, Valencia, CA). To verify the presence of a cloned insert, plasmid DNA in the pCR-4-TOPOH vector was digested using EcoRI and visualized on agarose gels. Automated sequencing of at least two clones from each PCR reaction was completed by Cleveland Genomics with primer M13 forward and reverse. Sequence data generated in the course of this work were deposited in GenBank. Additional sequences for other taxa used in the phylogenetic analyses were obtained from GenBank. Strain designations, GenBank accession numbers, and origin of strains are summarized in Table 1.

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0 Data analysis Complementary sequences were determined for the reverse sequences using Omiga2. Forward and complementary sequences were aligned using the CLUSTAL W Multiple Sequence Alignment Program (Thompson et al. 1994). Ambiguities and PCR errors were checked by eye, and the corrections according to the chromatograms were done where it was appropriate. Corrected sequences were aligned with Nostoc strains sequenced by other workers from Johansen’s laboratory (Table 1) as well as sequences from GenBank. In the first round of analyses, a large group of heterocytous taxa were examined to determine the phylogenetic position of our newly sequenced strains. We sampled 55 Nostocales which were named to species or which at least have been well studied and have appeared in previous phylogenetic analyses. We had two levels of outgrouping. To root the Nostocales we chose Microcoleus vaginatus Gomont, an oscillatorialean species with thylakoid structure similar to Nostocales. To root the 30 Nostocaceae without aerotopes (where our taxa belong morphologically), we used 21 Nostocaceae with aerotopes and 4 Microchaetaceae. Taxa were chosen to represent clades identified in other analyses with larger data sets (Lehtima¨ki et al. 2000; Iteman et al. 2002; Lyra et al. 2001; Rajaniemi et al. 2005). We excluded members of the Stigonematales since they are not closely related to our ingroup of interest. Finally, we chose taxa based on our ability to assess morphological features associated with the strains, such that we could conduct a total evidence parsimony analysis in our second round of analysis. Accession numbers for GenBank sequences used are as follows: Anabaena affinis (AY701541), A. augustumalis (AJ630458), A. crassa (AJ630413), A. cylindrica (AF091150), A. flos-aquae (AJ630420), A. kisseleviana (AY701557), A. lemmermanii (AJ630424), A. macrospora (AJ293115), A. planktonica (AJ630433), A. spiroides (AJ293116), Anabaenopsis sp. (AY038033), Aphanizomenon flos-aquae (AJ133154), Aph. issatschenkoi (AJ630446), Calothrix brevissima (AB074504), Coleodesmium wrangelii (AF334703), Cyanospira rippkae (AY038036), Cylindrospermopsis raciborskii (AB115489), Cylindrospermum stagnale (AJ133163), Microcoleus vaginatus (AF355362), Nodularia baltica (AJ133177), Nod. harveyana (AJ781146), Nod. sphaerocarpa (AJ133183), Nostoc sp. ATCC53789 (AF062638), Nostoc sp. Fin strain 152 (AJ133161), Nostoc sp. GSV224 (AF062637), Nostoc sp. Lobaria cyanobiont 34 (AF506259), Nostoc sp. PCC7120 (NC003272), Nostoc sp. SAG2028 (AJ344563), N. calcicola (AJ630447), N. commune (AY218833), N. edaphicum (AJ630449), N. ellipsosporum (AJ630450), N. entophytum (AB093490), N. linkia (AB074503), N. muscorum II (AJ630452), N. muscorum CENA61 (AY218828), N. piscinale (AY218832), N. punctiforme (AF027655), Rexia erecta (AY452533), Sprirestis rafaelensis (AF334692), Trichormus azollae (AJ630454), T. doliolum (AJ630455), T. variabilis HINDAK (AJ630456), T. variabilis GREIFWALD (AJ630457), Tolypothrix sp. (AB093486), Tol. distorta (AF334694). Sequence identity was compared among all strains, visually inspected using SeqEd, and aligned with other


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16S fragments using MacClade (Maddison & Maddison 1992). Sequences were analyzed using PAUP* 4.02b (Swofford 1998) to conduct heuristic searches (1000 replicates each) using neighbor-joining, parsimony, and maximum likelihood methods (Swofford 1998). Only the maximum likelihood trees of this round of analyses are reported herein. The maximum likelihood tree using the general time reversible model with corrected invariable sites (I) and gamma distribution shape parameters (G) using Modeltest v.3.06 (Posada & Crandall 1998) was constructed with 1000 rounds (each with its own random addition) of analysis. Bootstrap replicates were limited to 100 due to limited computing power. A similarity matrix for all strains was constructed using PAUP to compare sequence identity. In addition to the trees generated using molecular data alone, a total evidence cladogram was constructed using parsimony. This tree used the set of sequences that formed the basis for the first round of analyses. Random-additionsequence starting trees and tree-bisection-reconnection (TBR) was used to construct a 50% majority rule consensus maximum parsimony tree with a character set with combined morphological and sequence data. Morphological scoring is given in Table 2. Because detailed morphology was not known for all strains in the tree, the morphological characters were primarily those associated with genera. Published notes on the morphology of some strains made it possible to have variability recorded within genera, with presence or absence of gas vesicles being the most common intrageneric variable character. Tree support was subsequently assessed by running bootstrap analyses with 1000 replicates each. Increasing taxon sampling, even in related outgroups, has been found to greatly improve stability of phylogenetic analysis (Goertzen & Theriot 2003). Accordingly, an analysis using all available Nostocales and Stigonematales 16S rRNA sequences of length only somewhat smaller than our sequences (1115 bp) was run using maximum parsimony as an optimality criterion. For this tree we employed a heuristic search, initially with 100 random-addition replicates, steepest descent, nearest neighbor interchange (NNI) swapping, and MULPARS off. All trees in memory were then swapped with NNI and MULPARS on, followed by TBR swapping on all saved trees in memory and MULPARS off. Finally, all remaining trees in memory were swapped with TBR and MULPARS on. Bootstrap values were calculated from 100 replicates with one random addition sequence to jumble the data. Repeated blast searches using taxa from all clades in our initial trees (Figs 1, 2) were used to compile a file with all heterocytous taxa (many unnamed) available as of 1 September 2005 (a total of 244 taxa plus Oscillatoria sancta PCC7515 as a nonheterocytous outgroup taxon). Sequences were manually aligned by eye. Space does not permit listing the accession numbers of all strains used in this analysis. Secondary structures of conserved regions of the 16S-23S rRNA ITS regions were determined using RNA Mfold version 2.3 (Zuker 2003), with folding temperature set at 20uC. Short sequences of the ITS region were folded separately to avoid extensive ambiguity in structure. The D1-D19 helix was the most conserved structure, always

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Table 2. Data matrix of morphological characters scored for total evidence tree. A total of 14 morphological character states were scored as follows: (1) akinetes (0 5 absent, 1 5 present), (2) heterocytes (0 5 absent, 1 5 present), (3) colonial mucilage (0 5 absent, 1 5 present), (4) polarity of trichomes (0 5 isopolarity, 1 5 heteropolarity), (5) gas vesicles (0 5 absent, 1 5 present), (6) tapered trichomes (0 5 absent, 1 5 present), (7) intercalary production of heterocytes (0 5 absent, 1 5 present), (8) terminal production of heterocytes (0 5 absent, 1 5 present), (9) consistent fragmentation at the heterocyte (0 5 absent, 1 5 present), (10) oblique division giving rise to kinked filaments (0 5 absent, 1 5 present), (11) akinete position (0 5 absent, 1 5 apoheterocytic, 2 5 paraheterocytic), (12) cell division (0 5 in one plane only, 1 5 in two planes), (13) false branching (0 5 absent, 1 5 present), (14) end cell (0 5 rounded, 1 5 conical). Taxon

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Microcoleus vaginatus (outgroup) Anabaena augstumalis Anabaena cylindrica Anabaena (all other strains) Anabaenopsis sp. PCC9215 Aphanizomenon flos-aquae Aphanizomenon issatchenkoi Calothrix brevissima Coleodesmium wrangelii Cyanospira rippkae PCC9501 Cylindrospermopsis raciborskii Cylindrospermum stagnale Cylindrospermum sp. CENA 33 Mojavia pulchra Nodularia baltica BY1 Nodularia (all other strains) Nostoc sp. Fin 152 Nostoc (all other strains) Rexia erecta Spirirestis rafaelensis Tolypothrix distorta Tolypothrix sp. IAM M-259 Trichormus (all strains)

0 1 1 1 1 1 1 0 0 1 1 1 1 0 1 1 1 1 0 0 0 0 1

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 1 1 0 0 0 0 0

0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 1 1 0 0

0 0 0 1 1 1 1 0 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0

0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0

0 1 1 1 1 1 1 0 1 1 0 0 1 1 1 1 1 0 1 1 1 1 1

0 0 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 1 0 0 0 0 0

0 0 0 0 1 0 0 0 1 1 0 0 0 0 0 0 0 0 1 1 1 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

0 2 2 2 1 2 2 0 0 1 1 2 1 0 1 1 1 2 0 0 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 0

0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1

having the same (or very similar) beginning and ending base pairs, and it was possible to identify compensatory base pair changes in this helix (see Coleman & Mai 1997; Mai & Coleman 1997). The other helices (V2, Box B, V3) were identified by position in respect to the tRNA genes, the Box A, and terminus of the ITS but varied too extensively to identify compensatory base pair changes.

RESULTS AND OBSERVATIONS Nostoc lichenoides Vaucher (1803, Hist. D. Conferv. d’Eau Douce, p. 227) ˇ eha´kova´ et Johansen nom. validum R (Figs 3, 9, 12–17) DESCRIPTION: Colonies microscopic in the thallus of Collema tenax (SW.) Ach. on silty desert soil. On agar colonies starting as microscopic colonies but becoming a macroscopic thallus. Thallus begins as an aggregation of small, spherical to lobate colonies, which remain distinct for at least three months. Very old (6 months) macrocolonies fuse to produce a wrinkled, raised colony with a rough, broken margin along which small satellite colonies are evident. Macrocolonies are dark green for most of their life cycle, becoming yellowish brown when very old (6 months). Microcolonies are spherical to oblong to irregular, with trichomes densely arranged in a very firm and thin mucilage when the strain is first isolated. After passage of time, trichomes are loosely arranged in the mucilage and grow


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distant (2–10 mm) from the outer margin of the sheath. The sheath is colourless, even after exposure to sunlight. Only in very old cultures is the sheath yellowish. Trichomes distinctly constricted at the crosswalls, although the constriction is less evident in young trichomes (hormogonia). Hormogonia are rare, with cells more compressed and quadrate than trichomes in microcolonies, 2–4 mm in diameter. Vegetative cells are spherical to longer than wide, with a mean width of 3.5 mm and mean length of 4 mm, (2)3–5(7) mm wide, (2)3–5(6) mm long,. Heterocytes are spherical to elongated oval, usually intercalary and solitary, occasionally apical following trichome breakage, 3–6(7) mm long, 2–6(7) mm wide. Akinetes are rare, in series of up to 6, oval to rounded, with smooth or granular cell surface, intercalary, apoheterocytic, 4–9(10) mm long, 3–7 mm wide. Cell contents of all cells are non-granular, although some akinetes appear granular. HOLOTYPE: Botanical type designated by Drouet (1978, p. 211) from East Germany: Nordhausen, F.T. Ku¨tzing, in herbarium of Bornet and Flahault (PC) from Collema sp., determined by E. Bornet. REFERENCE STRAIN: Living strain CNP-AK1, deposited in the Culture Collection of Algal Laboratory in Trˇebonˇ, CZ, under accession number CCALA 699. CNP-AK1 isolated from Collema tenax by Anessa J. Kennedy from lichen specimen collected by Jayne Belnap in Canyonlands National Park, Utah, USA. Herbarium preparation of CNP-AK1 deposited in the Herbarium of Nonvascular Cryptogams, Brigham Young University, Provo, Utah: BRY C-37687.

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Figs. 1–11. Microphotographs of studied strains. Fig. 1. Bubbly, gelatinous macrocolony of Nostoc indistinguendum CM1-VF10. Fig. 2. Aggregation of spherical microcolonies of Nostoc desertorum CM1-VF14. Fig. 3. Wrinkled macrocolony of N. lichenoides CNP-AK1. Figs 4–8. Mojavia pulchra JT2-VF2, note aggregation of microcolonies (Fig. 4), hormocyte production (Figs 5, 7, 8), and compact arrangement of cells in microcolonies (Figs 6, 8). Figs 9, 10. Akinetes in N. lichenoides and N. indisinguenda, respectively. Fig. 11. Single trichomes and microcolony in N. indistinguendum.

ˇ eha´kova´ et Johansen sp. nova Nostoc indistinguendum R (Figs 1, 10, 11, 18–26) DESCRIPTIO: Coloniae in natura microscopicae, gelatinosae, despues macroscopicae in cultura, virides, cum margine levi, coloniae filiae not separantur; coloniae adultae cum bullis aeriis; coloniae sphaericae vel irregulares, cum trichomatibus dense implexis, despues confluentes, cum trichomatibus in muco laxe contortis. Vaginae semper incolores, 2–9 mm crassae. Trichomata circinata, ad dissepimentis constricta, 2–5 mm lata. Cellulae vegetativae


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longior quam latae, ovales vel quasi quadraticae vel cylindricae, contentu viridi, postea lutescente et granuloso, 2–5 3 2–5 mm. Heterocytae sphaericae ad elongate ovales, saepe terminales, rarissime intercalares, 3–7 3 2–6 mm. Akinetes sphaericae vel ovales, apoheterocyticae, intercalares, contentu granuloso, lutescente, 4–9 3 4–7 mm. Reproductio plerumque hormogoniis plus minusve rectis, cum cellulis subsphaericis vel irregulariter compressis. DESCRIPTION: Colonies are microscopic in desert soils. On agar, colonies are macroscopic and gelatinous at all stages of the life cycle, green in colour, with smooth margins, and

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Figs. 12–19. Drawings of two studied strains. Figs 12–17. N. lichenoides CNP-AK1. Fig. 12. Low-power view of aggregated microcolonies. Figs 13, 14. Free-living trichomes (from hormogonia). Figs 15–17. Young microcolonies. Figs 18, 19. N. indistinguendum CM1-VF10, filamentous and spherical forms of young colonies.

no satellite colonies. After three months, large air-filled bubbles form in the macrocolonies. Microcolonies are spherical to irregular, only observed in the first 60 days of the life cycle, becoming completely confluent after that time, with trichomes compact and indiscernible in the first three days, then becoming loosely arranged and growing distant (2–9 mm) from the outer margin of the sheath. The sheath is colourless, even after exposure to sunlight.


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Trichomes are curved and bent, constricted at the crosswalls, arranged throughout the mucilage, 2–5 mm wide. Hormogonia are common, fairly straight, with rounded or irregularly compressed cells. Vegetative cells are longer than wide, oval to quadratic or cylindrical with less evident constrictions at crosswalls, granular in older cells, 2–5(6) mm wide by 2–5(6) mm long. Heterocytes are spherical to elongated oval, usually apical, very rarely intercalary, 3–

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Figs. 20–26. N. indistinguendum CM1-VF10. Fig. 20. Low-power view of colony with dispersed trichomes. Figs 21–23, 25, 26. Colonies in various life cycle stages. Fig. 24. Hormogonia and free-living trichomes arising from hormogonia.

7 mm long by 2–6 mm wide, with the larger heterocytes in older colonies. Akinetes rounded or oval, intercalary, apoheterocytic, finely granular, yellowish, 4–9 mm long by 4–7 mm wide. HABITAT:

Subaerophytic in sandy desert soil.

TYPE LOCALITY: Clark Mountains, San Bernadino County, California, USA, 35u38.1059N latitude 115u30.7449W longitude, 1090 m in elevation.


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HOLOTYPE: BRY C-37689, Herbarium of Nonvascular Cryptogams, Brigham Young University, Provo, Utah. REFERENCE STRAIN: Collected 5 June 1998, by J.R. Johansen, isolated into pure culture by V.R. Flechtner, 1999. Living type CM1-VF10 deposited in the Culture Collection of Algal Laboratory in Trˇebonˇ, CZ, under accession number CCALA 692.

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REMARKS: Among the four strains characterized in this study, N. indistinguendum most closely resembles Nostoc desertorum but is distinguished from that species by having the loose, bubbly, mucilaginous colony; thinner akinetes; consistently colourless mucilage; and absence of compartmentalized mucilage in mature colonies. Morphologically, Nostoc indistinguendum was most similar to N. paludosum in cell size, arrangement of trichomes, and character of colonies. N. paludosum differs by having a yellowish sheath and akinetes with a smooth surface (Geitler 1932; Desikachary 1959). The ecology of both taxa is different as well. Nostoc paludosum was described from stagnant water, while N. indistinguendum is from desert soil. Molecular data are not available for N. paludosum. Nostoc indistinguendum is distinguished from N. commune by its consistently colourless sheath and microscopic colonies in nature. It is distinguished from N. edaphicum Kondratyeva by its larger heterocyte size and colony morphology.

ˇ eha´kova´ et Johansen sp. nova Nostoc desertorum R (Figs 2, 27–33) DESCRIPTIO: Coloniae microscopicae in natura, in cultura primo minutae, sphaericae vel lobatae, agglomeratae, in agglomerationes macroscopicae, despues teges planas formantes, atrovirides, adultae ad luteovirides vel griseovirides. Vaginae tenues, firmae, incolores, minus quam 4 mm latae. Trichomata valde circinata in coloniis juvenalibus, despues circinata, valde constricta ad dissepimentis, 2–7 mm lata. Cellulae vegetativae subsphaericae vel irregulariter complanatae, barriliformes ad ovales, plus minusve quadraticae post divisione, 2–7 3 2–7 mm. Heterocytae sphaericae vel ovales, solitariae, intercalares vel terminales, 3–8 3 2–8 mm. Akinetes subsphaericae vel ovales, seriebus ordinatae, intercalares vel apicales, episporio lutescenti, superficie levi vel granuloso, 6–11 3 5–11 mm. Reproductio hormogoniis cum cellulis ovalibus vel quadraticis, complanatis, 2–6 mm longis, 2–8 mm latis. DESCRIPTION: Colonies are microscopic and free-living in desert soils. On agar, macroscopic colonies start as conglomerations of small, spherical to lobate colonies, which remain distinct throughout the life cycle. Even old colonies consist of aggregations of microcolonies, and form fairly flat masses on the agar surface (Fig. 7B). Macrocolonies start dark green, and become yellow-green to gray green. Microcolonies are rounded, irregular, not becoming macroscopic, averaging 40–45 mm long by 30–35 mm wide. Sheaths are thin, firm, and colourless in young colonies, with trichomes lying less than 4 mm from the outer surface, becoming brownish yellow and compartmentalized in older colonies. Trichomes are unrecognizable in the younger colonies, becoming visible but still very compact after one month of growth, curved and bent, 2–7 mm wide. Hormogonia with oval to quadratic compressed cells are 2–6 mm long by 2–5 mm wide. Vegetative cells are rounded to irregularly compressed in compact colonies before trichomes are visible; in the trichome they are usually barrelshaped to oval, or quadratic after division, constricted at the cross-walls, 2–7 mm long by 2–7 mm wide. Heterocytes


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are oval to rounded, intercalary or apical in position, 3– 8 mm long by 2–8 mm wide. Akinetes are round to oval, intercalary to apical in position, with smooth to granular surface, yellowish, 6–11 mm long by 5–11 wide. HABITAT:


TYPE LOCALITY: Clark Mountains, San Bernadino County, California, USA, 35u38.1059N latitude 115u30.7449W longitude, 1090 m in elevation. HOLOTYPE: designated BRY C-37691, Herbarium of Nonvascular Cryptogams, Brigham Young University, Provo, Utah. REFERENCE STRAIN: Collected 5 June 1998, by J.R. Johansen, isolated into pure culture by V.R. Flechtner, 1999. Living type CM1-VF14 deposited in the Culture Collection of Algal Laboratory in Trˇebonˇ, CZ, under accession number CCALA 693. REMARKS: As stated above, Nostoc desertorum most closely resembles N. indistinguendum but is distinguished by both morphological and molecular (ITS) characters (see below). Of previously described species, N. desertorum most closely resembles N. edaphicum, N. punctiforme, and N. commune. It also shows similarities to N. microscopicum Born. & Flah. and N. muscorum. However, it does not match any of these taxa. Nostoc muscorum, N. microscopicum, and N. commune create macroscopic colonies observable without microscopy, and all have yellow to brown sheaths (Geitler 1932), whereas N. desertorum is only microscopic in nature and has a colourless sheath throughout most of its life cycle. Shape and dimension of vegetative cells, heterocytes, and akinetes were similar among all these taxa and is generally not very informative within the genus. Nostoc edaphicum is similar in both ecology and morphology but has notably smaller heterocytes (Table 4). Nostoc punctiforme is microscopic like N. desertorum and also lacks the brownish mucilage. However, habitat specificity and molecular characteristics are quite different. The N. punctiforme strain sequenced (ATCC 29133) is from the roots of Macrozamia, a cycad from Australia. Nostoc desertorum has a very different structure in the ITS, as will be shown below.

ˇ eha´kova´ et Johansen gen. nov. Mojavia R DESCRIPTIO: Genus cyanoprocaryoticum generis Nostoc similis. Coloniae microscopicae in natura, subsphaericae, ellipsoideae vel irregulares, tegumentis firmis et mucilaginosis circumdatae, despues in seriebus macroscopicis, repentis vel erectis ordinatae in vitro; series subcoloniis pseudofilamentosae, de subcoloniis et microcoloniis compositae, praecipue in vitro. Cellulae subsphaericae, in coloniis in trichomas uniseriatas vel pluriseriatas et valde flexuosas conjunctae, ad dissepimenta valde constrictae, vel in aggregationis densis agglomeratae. Heterocytae semper terminales, vel laterales in agglomerationis, heterocytae intercalares raris. Coloniae iuvenes cum heterocytis utrinque terminalibus. Reproductio disintegratione coloniis, cellulis solitariis liberantis de coloniis, vel hormogoniis paulocellularibus. Hormogoniae germinantes sine hetero-

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Figs. 27–33. Nostoc desertorum CM1-VF14. Fig. 27. Low-power view of colony with dispersed trichomes. Fig. 28. Older colony showing compartmentalization of colonial mucilage. Figs 29, 33. Young colonies. Figs 30–32. Free-living trichomes with intercalary heterocytes and hormogonial stages.

cytis, saepe cum cellulis binis dilatatis intercalaribus; hormogoniae plerumque acute flexae inter cellulis dilatatis. DESCRIPTION: Cyanobacterial genus similar to the genus Nostoc. Colonies microscopic in nature, subsphaerical, ellipsoid to irregular, surrounded by a firm and mucilag-


9

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inous covering. Series of subcolonies create macroscopic, upright or horizontal pseudofilaments in liquid culture. Cells subsphaerical, joined into uniseriate trichomes or strongly flexuous pleuroseriate colonies, strongly constricted at the crosswalls, or agglomerated into dense aggregations. Heterocytes mainly terminal, or lateral in

0


agglomerations, with intercalary heterocytes very rare. Young colonies only with terminal heterocytes. Reproduction with colonial disintegration, by free solitary cells (hormocytes) or multicelled hormogonia. Hormogonia germinating without heterocytes, often with intercalary paired widened cells which often cause acute bending of the hormogonia. TYPE SPECIES OF THE GENUS:

Mojavia pulchra.

ˇ eha´kova´ et Johansen sp. nov. Mojavia pulchra R (Figs 4–8, 34–43) DESCRIPTIO: Coloniae in natura edaphicae, iuvenes microscopicae, despues macroscopicae in cultura, sphaericae, irregulariter seriatum dispositae, seudofilamentosae, erectes, atrovirides, adultissimae luteo-fuscae. Trichomata valde contorta et fragmentosa in coloniis innenilibus, in coloniis adultis curta, irregulariter flexuosa. Vaginae mucilaginosae tenues, firmae, incolorae ad luteo-fuscae, ad 1–2 mm ab cellulis distantae. Cellulae vegetativae subsphaericae vel barriliformes, plus minusve isodiametricae, 4–10(12) mm longae, 4–10 (11) mm latae, contentum viride-fusci. Heterocytae ovales vel subsphaericae, intercalares vel terminales, 4–10 (12) mm longae, (2) 4–8 (10) mm latae. Akinetes not observantur. Reproductio hormogoniis raris, cum cellulis ovalibus vel curtis, barriliformibus, circu, 10 mm longis et 7 mm latis. DESCRIPTION: Colonies are microscopic in the desert soil. On agar, colonies do not grow very well. They grow better in liquid medium. In liquid they start as flat conglomerations of very small spheres and remain in this state for 2– 3 months, after which they create erect filaments (Fig. 11A, B). Macroscopic colonies are dark green for most of the life cycle, becoming yellowish-brown when very old (6 months). Microcolonies filamentous, spherical or irregular, arranged in pseudofilaments or irregular aggregations (Figs 7D–H, 11A–D, I, J). The sheath around the microscopic colonies is firm, thin, and colourless at the beginning of the life cycle (Fig. 7F) and later yellowish brown (Fig. 7E, G, H). Vegetative cells grow within 1–2 mm from the outer margin of sheath. Trichomes are unrecognizable in the microscopic colonies throughout the entire life cycle (Fig. 7D–H). Hormogonia are very rare, with oval or compressed cells. The dimensions of the cells of hormogonia are 10 mm long, 7 mm wide. Hormocytes released from very old aggregations of microcolonies with compartmentalized mucilage, giving rise to trichomes or microcolonies. Vegetative cells are rounded to irregularly compressed in the compact colonies, 4–10 (12) mm long, 4–10 (11) mm wide. The colour of vegetative cells is greenish brown. Heterocytes oval to rounded, apical in position or lateral in microcolonies (Fig. 7F), 4–10 (12) mm long, (2) 4–8 (10) mm wide. Akinetes were not observed during 3 years of our observation. HABITAT:


TYPE LOCALITY: Cadiz Valley, Joshua Tree National Park, San Bernadino County, California, USA, 34u06.5929N latitude 115u28.8219W longitude.


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HOLOTYPE: Designated BRY C-37693, Herbarium of Nonvascular Cryptogams, Brigham Young University, Provo, Utah. REFERENCE STRAIN: Collected 8 June 1998 by J.R. Johansen, isolated into pure culture by V.R. Flechtner, 1999. Living type JT2-VF2 deposited in the Culture Collection of Algal Laboratory in Trˇebonˇ, CZ, under accession number CCALA 691. REMARKS: This species has several distinctive morphological characters, including both the acute V-shaped bending of the filament and the unique filamentous colony structure. Its recognition as a new genus, however, is only weakly justified by morphological differences. The basis for recognition is its place in molecular phylogenies and the distinctive secondary structure of the 16S-23S ITS region, differences which will be discussed below.

Phylogenetic analyses Distance, parsimony, and maximum likelihood analysis based on 16S rRNA sequence data for only 55 Nostocales yielded trees with nearly identical topology. We show only the maximum likelihood tree to represent these analyses (Fig. 44). According to all three of these analyses, a clade containing most of the Nostoc taxa was evident. Taxa falling outside this clade included Nostoc piscinale CENA21, Nostoc muscorum CENA61, Nostoc sp. Fin 152, and Mojavia pulchra. In order to include these taxa in the Nostoc clade, we would have to consider Trichormus azollae (Strasb.) Kom. et Anagnost., Cylindrospermum stagnale Born. & Flah., Cylindrospermum sp. CENA33, Calothrix brevissima W. & G.S. West, and Tolypothrix sp. IAM M-249 to all be Nostoc. Nostoc commune appears to be polyphyletic. The UTEX584 strain was isolated from Scotland by T. Gibson, but no additional details about the strain are given on the UTEX website. The type specimen for N. commune was macroscopic and isolated from central Europe. Our strain of N. commune (EV1-KK1) was isolated from field soil in the Czech Republic, was macroscopic, and fitted the description of N. commune in every detail. Given that UTEX 584 is consistently sister to a lichen phycobiont in these three analyses, we question the identity of this strain with N. commune. Nostoc lichenoides, CNP-AK1, the phycobiont from Collema tenax, has been previously identified as N. commune, N. punctiforme, and N. sphaericum Vauch. ex Born. et Flah. Its placement outside the clades containing these taxa dictates that it is not in any of these species. The clade containing Nostoc sp. GSV224, N. punctiforme, N. commune, N. desertorum, and N. indistinguendum is a monophyletic cluster in every analysis (Fig. 44, clade A). Thus, we conclude that the new taxa, if justified, must be diagnosable from N. punctiforme and N. commune. Comparisons of the new species to N. muscorum and N. edaphicum are of interest, but we conclude that the desert strains in the N. commune clade cannot be placed in species falling outside that clade. In the trees based on molecular data alone (for 55 taxa), there is a cluster of strains that clearly forms a monophyletic

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Figs. 34–43. Mojavia pulchra JT2-VF2. Figs 34, 35. Pseudofilamentous aggregations of microcolonies (scales 5 10 mm). Figs 36, 37. Filaments with terminal heterocytes. Figs 38–41. Structures arising from hormocytes, showing specialized pairs of widened cells, which cause acute bending of the trichome (arrows). Figs 42, 43. Microcolonies.

group with strong (100%) bootstrap support (Fig. 44, clade C). A larger cluster apparently is monophyletic (Fig. 44, clade D) but lacks support. The total evidence tree could only be examined with parsimony as an optimality criterion. This tree (Fig. 45) had


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strikingly similar topology to the trees based on molecular sequence data only (Fig. 44, several trees not illustrated). Mojavia still clusters with Tolypothrix sp. IAM M-259 and is clearly outside of the Nostoc clade (Fig. 45, clades C or D, with taxa identical to Fig. 44, clades C or D, respectively).

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Fig. 44. Topology of 16S rRNA gene sequence maximum likelihood tree with bootstrap values above 50% reported. Taxa sequenced for this study in boldface font. Clade ‘‘C’’ is Nostoc sensu stricto; see text for explanation of other labeled clades.

Fig. 45. Topology of the total evidence parsimony tree with bootstrap values above 50% reported. Taxa sequenced for this study in boldface font. Clade ‘‘C’’ is Nostoc sensu stricto; see text for explanation of other labeled clades.

The taxa in Fig. 45, clade A, are the same as those in Fig. 44, clade A. However, the total evidence tree differs from the ML tree in that Nostoc lichenoides and its associated taxa form a distinct clade. The placement of Nostoc commune UTEX 584 also changes in the total evidence tree (Fig. 45). Addition of the morphological criteria did resolve the Microchaetaceae (Spirirestis, Tolypothrix, Coleodesmium, Rexia) but did little to resolve other unresolved nodes in the maximum likelihood tree. The tree containing all available heterocytous taxa with a 1115-bp segment of the 16S rRNA gene (Fig. 46) differed slightly in topology from the trees based on less taxon sampling (Figs 44, 45). Addition of taxa in the Anabaena/ Aphanizomenon/Nodularia clade decreased bootstrap support for this node but did not substantially change its topology. The Microchaetaceae (4 taxa) were sister to the clade containing the Stigonematales (25 taxa) and may have helped to resolve the clades containing the Nostocaceae. Importantly, this tree still showed high integrity in the Nostoc sensu stricto clade (Fig. 46, clade C), which contains all strains identified as N. commune, the generitype of the genus. Mojavia is sister to Nostoc sensu stricto, and the clade containing Trichormus variabilis (Ku¨tz. Ex Born. & Flah.) Kom. & Anagn., Trichormus azollae (Strasb.) Kom. & Anagn., and PCC7120 is sister to the clade containing both Mojavia and Nostoc. The other Nostoc group, outside clade C in the other two analyses (N. entophytum Born. &

Flah., N. linkia (Roth) Born.et Flah., N. muscorum II, N. ellipsosporum (Desm.) Rabenh. V, etc.), was also outside clade C in this analysis (Fig. 46, Nostoc Group II). Nostoc lichenoides and N. desertorum were in the Nostoc sensu stricto clade in a position sister to other lichen photobiont Nostoc. Nostoc indistinguendum was not resolved as a monophyletic group with those taxa we designate as N. cf. indistinguendum as it was in the analyses with fewer taxa, although all fell out as a metataxon at the base of the true Nostoc.


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Evidence of evolutionary divergence: 16S rRNA sequence dissimilarity Dissimilarity of 16S rRNA sequences above 0.025 has often been used as evidence of evolutionary divergence and is a criterion for species recognition among many who study microbial taxonomy. We consider the 0.025 cutoff point to be arbitrary and the use of sequence similarity to be a phenetic and therefore questionable approach. However, high sequence dissimilarity does represent one line of evidence of evolutionary divergence and therefore is worthy of note. We compared sequence dissimilarity among all taxa in clade B (Fig. 44). Nostoc desertorum had greater than 0.025 dissimilarity from all four strains of N. indistinguendum (0.027–0.032). Also, N. desertorum had high dissimilarity

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Fig. 46. Topology of the maximum parsimony tree conducted with an extended set of heterocytous taxa (245 taxa, 1115 bp of the 16S rRNA gene), with bootstrap values above 50% reported. The Anabaena/Aphanizomenon/Nodularia clade included these genera as well as Anabaenopsis, Cyanospira, Cylindrospermopsis, and two strains assigned to Trichormus variabilis (the Greifswald and Hindak strains, see Figs 1, 2). The soil Nostoc group included N. edaphicum, N. calcicola, N. punctiforme, N. commune, and several Peltigera and Nephroma photobionts. The Trichormus clade included T. azollae, T. variabilis, PCC7120, and three unidentified taxa. Nostoc Group II included N. ellipsosporum, N. entophytum, N. linkia, and N. muscorum and three unidentified taxa. The clade labeled as ‘‘C’’ contained all taxa contained in the clade ‘‘C’’ of Figs 1 and 2 as well as additional taxa used in this analysis.

with strains of N. lichenoides (0.024–0.031). Nostoc indistinguendum and N. lichenoides strains had a dissimilarity range of 0.015–0.031. All other taxa in clade B (Fig. 44) had lower comparative dissimilarity, and we consider dissimilarity to be uninformative in these instances. The high 16S dissimilarity does provide evidence of divergence of N. desertorum from the other two desert taxa mentioned. Mojavia pulchra was most similar to Tolypothrix sp. IAM M-259 (dissimilarity 5 0.030). It was thus a separate species from all sequenced taxa in GenBank to date based on the dissimilarity criterion (the 0.025 cutoff). It stood alone in a position sister to Nostoc in the third analysis (Fig. 46).


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Evidence of evolutionary divergence: ITS secondary structure The 16S-23S ITS region in Nostoc was highly variable. We observed ITS regions with both tRNA genes, no tRNA genes, and one tRNA gene and with an unusual ITS with a deletion between the middle of tRNAIle and tRNAAla to produce a nonsense region containing two contiguous fragments of the beginning of tRNAIle and the end of tRNAAla (possibly a hybrid dimer?). We encountered up to four different types of operon in some of our strains (both tRNAs, fragmented tRNAs, and two different ITS types with no tRNAs). It is very difficult to align these ITS regions (Iteman et al. 2000), although there are several semiconserved secondary structures that can be compared.

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Figs. 47–58. Secondary structure of D1-D19 helices from the 16S-23S ITS region, including multiple patterns displayed in multiple intragenomic operons. Figs 47, 48. N. lichenoides CNP-AK1, operons with no tRNA and both tRNA genes, respectively. Fig. 49. Nostoc sp. JT1-VF3, operon with one tRNA gene. Figs 50, 51. N. indistinguendum CM1-VF10, operons with both tRNA and no tRNA genes, respectively. Fig. 52. Nostoc cf. indistinguendum FI5-VF12, operon with one tRNA gene. Fig. 53. Nostoc cf. indistinguendum FI5-VF1, operon with no tRNA genes. Fig. 54. Nostoc cf. indistinguendum FI5-VF4, operon with both tRNA genes. Fig. 55. N. desertorum CM1VF14, operons with both and no tRNA genes. Fig. 56. N. punctiforme ATCC29133, operon with both tRNA genes. Fig. 57. N. commune EV1-KK1, operon with no tRNA genes. Fig. 58. Mojavia pulchra JT2-VF2, operon with no tRNA genes.

These include the D1-D19 helix, the V2 helix (only in operons with 2 tRNA genes), the Box B helix, and the V3 helix. There are other conserved regions in the ITS (D2, D3, Box A, D4) that have no structural changes, and the D5 helix, which can be determined with confidence only if the 23S-5S ITS sequence is known. We found several different patterns in the D1-D19 helix, Box B, and V3 regions of the ITS. The D1-D19 helix was the most conserved structure, although the sequence varied considerably. All Nostoc taxa for which ITS sequences were available had a one-sided loop at the base of the stem, one or sometimes two small loops above that structure, then a large loop topped by a small, 3-bp stem and loop end


14

(Figs 47–58). A unique D1-D19 helix was present in Mojavia pulchra (note that only 2 bp separate the top loops, the C-UU internal loop has lost a U, and several unique compensatory base pair changes are present in the central region of the helix, Fig. 58). Secondary structure of members of the Microchaetaceae (Rexia, Spirirestis, Tolypothrix, Coleodesmium) likewise have only 2 bp between the top two loops but have a very different sequence structure in the terminal loop as well as different sequence in the main helix. The V2 regions varied slightly but were similar in structure and length (67–76 bp). They were very similar to those found in Nostoc sp. PCC7120 (Iteman et al. 2000).

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Figs. 59–70. Secondary structure of Box-B region of 16S-23S ITS region, including multiple patterns displayed in multiple intragenomic operons (same operons as in Figs 47–58). Figs 59, 60. N. lichenoides CNP-AK1. Fig. 61. Nostoc sp. JT1-VF3. Fig. 62. N. indistinguendum CM1-VF10. Fig. 63. Nostoc cf. indistinguendum FI5-VF12. Fig. 64. Nostoc cf. indistinguendum FI5-VF1. Fig. 65. Nostoc cf. indistinguendum FI5-VF4. Figs 66, 67. N. desertorum CM1-VF14. Fig. 68. N. punctiforme ATCC29133. Fig. 69. N. commune EV1-KK1. Fig. 70. Mojavia pulchra JT2-VF2.

The Box B helix was more variable (Figs 59–70) in structure, having different numbers of internal loops (some centered, some forming on the side) and variable lengths (40–55 bp). The only constant was that it was always an unbranched helix. There was intragenomic variability among operons of the same strain, and this was more pronounced than the variability observed in the D1-D19 helices. The V3 was the most variable helix, varying in length from 32 to 110 bp. It was consistently an unbranched helix. The base of the helix was also conserved in sequence, and recurrent patterns could be seen in some of the loops. Taxa that were similar in ecology or morphology often showed consistencies in their structure. For example, N. punctiforme ATCC 29133 and N. lichenoides CNP-AK1 had exceptionally long V3s, with similar placement and size of loops (Figs 79, 71). The other V3s were much shorter (36–50 bp) and differed widely in structure (Figs 72–78, 80). Intragenomic variability among operons was high in N. lichenoides (Figs 71, 72). The V3 of Mojavia was long but quite distinct from the V3 helix in all other taxa (Fig. 81). At least some differences in at least some of the conserved structures in the ITS region were present among all strains (no two strains were identical in ITS). While these could be considered evidence of evolutionary divergence, at this time it is difficult to assess the importance of the differences observed, particularly with the problem of multiple operons that we suspect were undersampled. Evidence of evolutionary divergence: morphology In nature, N. desertorum, N. indistinguendum, N. lichenoides, and Mojavia pulchra were microscopic. Of the four desert species isolated from desert soils, the Nostoc taxa grew very well on agar-solidified N-free Z-8 medium. Mojavia pulchra grew better in liquid N-free Z-8 medium. All observed strains created macroscopic colonies in


15

laboratory conditions. Colonies differed in morphology, and the character of colonies on agar or in liquid was characteristic for each species (Table 4). The clustering of many microcolonies in M. pulchra into large pseudofilaments was a very distinctive autapomorphic feature. The dimensions of the vegetative cells, heterocytes, and akinetes differed significantly among strains (Table 3). Comparisons of length and width of vegetative cells and heterocytes were made for all four reference strains, whilst for akinetes this was done only for the three Nostoc strains, as akinetes were not observed in M. pulchra (Table 4). The length of akinetes did not differ significantly among the Nostoc strains, although N. desertorum did have a considerably higher range of akinete sizes (up to 11 mm wide by 11 mm long). Mojavia pulchra was very different from the other three strains (Table 3). This strain had significantly longer and wider vegetative cells and heterocytes than the Nostoc strains. The differences in dimensions among the three Nostoc strains were small in magnitude (Table 3). Evidence of evolutionary divergence: habitat specificity Taxa described from Europe that are similar in some way to our four taxa include N. punctiforme, N. edaphicum, N. commune, N. paludosum, N. muscorum, and N. microscopicum (Table 4). However, all of these taxa were described from biotopes very different from desert soil. We consider habitat specificity to be a taxonomically informative character and on this basis alone would separate our taxa from the species originally described from Europe.

DISCUSSION Recently, there have been a number of papers on ecophysiology, biochemistry, and cell structure of Nostoc

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Figs. 71–81. Secondary structure of the V3 region of the 16S-23S ITS region, including multiple patterns displayed in multiple intragenomic operons (same operons as in Figs 47–58). Figs 71, 72. N. lichenoides CNP-AK1. Fig. 73. Nostoc sp. JT1-VF3. Fig. 74. N. indistinguendum CM1-VF10 (all operons). Fig. 75. Nostoc cf. indistinguendum FI5-VF12. Fig. 76. Nostoc cf. indistinguendum FI5-VF1. Fig. 77. Nostoc cf. indistinguendum FI5-VF4. Fig. 78. N. desertorum CM1-VF14. Fig. 79. N. punctiforme ATCC29133. Fig. 80. N. commune EV1-KK1. Fig. 81. Mojavia pulchra JT2-VF2.

spp. (Mollenhauer 1970, 1985a, b, 1986a, b; Potts 1994, 2004; Dodds et al. 1995; Hrouzek et al. 2003; Becker et al. 2004; Rajaniemi et al. 2005). For the most part, these papers do not propose taxonomic changes, although some give morphological characterizations (Hrouzek et al. 2003). Mollenhauer et al. (1994, 1999) studied the morphology of the macroscopic species within Nostoc and provide insightful comments about approaching problems within the genus. We agree with their assessment that the microscopic species in the genus are understudied, poorly known, and likely represent many more species than currently described (Mollenhauer et al. 1994). Kondratyeva and Kislova (1992, 2002) determined that life cycles of Nostoc are complex.

They questioned the value of the density of trichomes to separate species, as this trait varies even within cultures. Our observations are consistent with their findings. Baker et al. (2003) concluded that the cyanobiont (PCC7120) in Azolla was neither Nostoc nor Anabaena based on sequence data of the V6-V8 region of the 16S rRNA gene. Our data support the placement of PCC7120 in a clade containing both Trichormus variabilis and T. azollae (Trichormus clade, Fig. 3). Two other strains have been assigned to Trichormus (T. variabilis Greifswald and Hindak strains, Figs 44, 45, in Fig. 46 as part of Anabaena/ Aphanizomenon/Nodularia clade), but they fall outside the Trichormus clade. Nostoc sp. Fin strain 152, a toxic strain

Table 3. Mean cell dimensions 6 s for vegetative cells, heterocytes, and akinetes. P values for analysis of variance (ANOVA) are given. Those means not sharing letter superscripts are significantly (a 5 0.05) different according to Tukey’s HSD test. CNP-AK1 Feature measured Vegetative cells: length Vegetative cells: width Heterocytes: length Heterocytes: width Akinetes: length Akinetes: width

CM1-VF10

Nostoc lichenoides 4.05 3.50 4.32 4.08 6.38 4.48

6 6 6 6 6 6

Nostoc indistinguendum

b

0.04 0.04a 0.05a 0.05b 0.12a 0.10c


3.80 3.45 4.18 3.91 6.09 5.67

16

6 6 6 6 6 6

a

0.04 0.05a 0.05a 0.06ab 0.11a 0.10a

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CM1-VF14 Nostoc desertorum 4.01 3.75 4.11 3.72 6.01 5.24

6 6 6 6 6 6

b

0.03 0.04b 0.05a 0.06a 0.15a 0.12b

JT2-VF2

ANOVA

Mojavia pulchra 6.52 5.26 7.08 5.57

6 6 6 6

c

0.07 0.06c 0.09b 0.08c

P value P P P P P P

, , , , 5 ,

0.01 0.01 0.01 0.01 0.079 0.01

clusters of microspheres in sheets or filaments small spheres, to gelatinous small and irregular small and irregular lobate or foliate rounded to ellipsoidal rounded to flat sheet

M. pulchra JT2-VF2

N. edaphicum

N. punctiforme N. commune

N. microscopicum

N. muscorum

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rounded to oval oval to rounded barrel-shaped spherical to conical rounded rounded rounded rounded

N. commune

N. microscopicum N. muscorum

7 6–7

4–6.5 (6–10) 7

1.8–3.7

3–7

rounded to oval

Length

N. indistinguendum CM1-VF10 N. desertorum CM1-VF14 M. pulchra JT2-VF2 N. paludosum N. edaphicum

Shape

7 6–7

4–6.5 (6–10) 7

4–10 3–3.5 2.1–3.9

2–8

2–6

2–6

smooth

rounded

oval oval

rounded

smooth smooth

smooth smooth, yellow

smooth

granular, yellowish smooth, yellowish

smooth, granular

Surface

Akinetes

microscopic to 0.5 mm microscopic to 0.5 mm microscopic macroscopic, several cm macroscopic, up to 1 cm macroscopic, up to 5 cm

microscopic

microscopic

microscopic microscopic

Size in nature

rounded to oval rounded to oval rounded to oval not observed oval oval

Shape

olive green to yellow brown

olive green to brown

deep blue green olive green to brownish

blue green to yellowish

dark green to deep brown blue green to golden

Width

Dimension

Heterocytes

3–6

N. punctiforme

dark green dark green

Colour

at first small spheres, later flat green to yellow green

clustered spheres gelatinous, bubbly

Shape

N. lichenoides CNP-AK1 rounded to oval

Species

N. paludosum

N. lichenoides CNP-AK1 N. indistinguendum CM1-VF10 N. desertorum CM1-VF14

Species

Phycologia phya-46-04-10.3d 2/5/07 10:19:58 Macrocolony on agar

9–15 8–12

5–8 (10–20) 4.5–6

6.8 1.8–5.7

6–11

4–9

4–9

Length

6–7 4–8

6.5 (10–20) 4.5–6

4–4.5 2.4–5.6

5–11

4–7

3–7

Width

Dimension

yellow brown

yellow

colourless to yellowish colourless to yellowish colourless yellow brown

colourless to brown

colourless

colourless to yellow colourless

Sheath colour

Ecology

3–6

3–3.5

4–10

2–7

2–7 2–6

Width

6–8

5–8

3.5–4

5–8

3–4 (5–6) 3–4 (5–6) 4.5–6 4.5–6

4–10

2–7

2–6 2–6

Length

humid soil, or cycad tubercules (larger size 5 Meeks et al. 2001) periodically dry soil in humid environments, cosmopolitan wet soil among mosses and rocks wet soil among mosses and rocks, cosmopolitan

soil, Mojave Desert stagnant water, cosmopolitan salty soil, semiarid to temperate climates

soil, Mojave Desert

soil, Mojave Desert

phycobiont of Collema tenax from semiarid steppe

dense, R

dense, R

very dense, U dense, U to R

dense, R

dense, U in small colonies, R in larger ones dense, U except for hormogonia loose, always R

compact, U, to very loose, R loose, R

Arrangement of trichomes

Vegetative cell dimensions

Table 4. The comparison of morphological characteristics of the strains studied and similar species described in Geitler (1932), Desikachary (1959), Starmach (1966), Hrouzek et al. (2003), and Meeks et al. (2001). Abbreviations: R 5 trichomes are recognizable in the mucilage of microcolony, U 5 trichomes are unrecognizable in the mucilage of microcolony.

0

0


from a lake in Finland (Rouhiainen et al. 1995; Lyra et al. 1997), also falls well outside the Nostoc clade according to all our analyses. Molecular evidence is making it increasingly clear that Nostoc is a polyphyletic assemblage as currently morphologically circumscribed. This is supported by studies of the 16S rRNA gene (Rajaniemi et al. 2005; this study), as well as analyses using nifD (Henson et al. 2004), nifH (Tamas et al. 2000), and rbcLX (Rajaniemi et al. 2005). Nostoc as defined morphologically is monophyletic according to molecular evidence only when taxon sampling is very small (Henson et al. 2002; Gugger & Hoffmann 2004). Our study demonstrates the need for greater taxon sampling among nostocoid strains. This is underway in other laboratories (Ventura & Hrouzek, personal communication). Other workers have suggested using tRNA (UAA) group I intron DNA sequence to differentiate Nostoc (Wright et al. 2001), but this gene shows little variation among N. commune populations (Novis & Smissen 2006). The importance of maximizing taxon sampling is clearly demonstrated in Goertzen and Theriot (2003). All trees we generated (many not discussed in this paper) shared the following features of topology: (1) The Nostoc clade containing N. commune always maintained its integrity. It always included the lichen phycobionts as well as terrestrial representatives of Nostoc, and it always excluded species in other genera, Nostoc Fin 152, and Mojavia. (2) Aerotopeproducing members of the Nostocaceae formed a monophyletic group (3) Mojavia occupied an unresolved position near the base of Nostoc sensu stricto, generally among Trichormus, Calothrix brevissima, Tolypothrix IAM M249, and some aquatic Nostoc. and (4) Nostoc lichenoides formed a monophyletic group. The tree with greater taxon sampling (Fig. 46) differed in some minor respects: (1) N. indistinguendum was a basal metataxon rather than a clear monophyletic group, (2) N. desertorum was sister to N. lichenoides instead of N. indistinguendum, and (3) the base of the Nostocales was much better resolved through the addition of the Stigonematales. We have used a polyphasic approach to the taxonomy of Nostoc from desert soil and the comparison of those species with previously described Nostoc species catalogued by Geitler (1932), Desikachary (1959), Kondratyeva (1968), and Mollenhauer et al. (1999). Exact determinations of Nostoc species are not easy, especially from desert soils where most are consistently microscopic. Forty-two Nostoc species were considered valid and described in Geitler (1932) with 23 species recognized in Desikachary (1959), but none were described from, or even subsequently attributed to, desert soils. Only N. flagelliforme Bornet & Flahault (1886–1888) was originally described from warm temperate arid soils. A total of 210 species of Nostoc were mentioned in Drouet (1978). Many of them, however, are nomenclaturally invalid. It is possible that some taxa belong within the variation range of other species or represent ecophenotypes or developmental stages of a longer life cycle. However, it is also likely that morphologically similar but ecologically and genetically different species have been wrongly combined in inadequately described, broadly defined morphospecies (Casamatta et al. 2003). The assertion of many authors that the majority of


18

cyanophyte species have a cosmopolitan distribution arises also mainly from misidentifications (Koma´rek 1985; Casamatta et al. 2005). 16S rRNA sequence data have been used for a number of years to examine phylogenetic relationships of cyanobacteria at all taxonomic levels (Wilmotte et al. 1992; Turner 1997; Wilmotte & Herdman 2001). The 16S rRNA gene is especially valuable for phylogenetic study because it has highly conserved regions (for assessment of distant relations) as well as some variable regions (for assessment of closer relations). Although analysis of the 16S rRNA gene appears consistently useful for resolution of higherlevel taxonomic relationships, it is not always useful for determination of species. Stackebrandt and Goebel (1994) found that when two strains have 16S rRNA genetic identity below 97.5%, they consistently have DNA-DNA hybridization values below 70%, which has been used as a firm criterion for recognizing bacterial species (Wayne et al. 1987). Unfortunately, when 16S rRNA sequences are more than 97.5% similar, one cannot assume that the DNA-DNA hybridization levels are higher than 70%. Bacterial strains have been found that are 99.8% similar in their 16S rRNA and yet are still well below the 70% DNA-DNA hybridization level (Lachance 1981; Stackebrandt & Goebel 1994). In fact, 16S rRNA sequence identity may not be sufficient to guarantee bacterial species identity (Fox et al. 1992). No relationships between 16S rRNA and generic identity have been postulated formally, but within the Oscillatoriales different genera are typically less than ,95% similar, while in the Nostocales different genera have been found to be as similar as 99% in their 16S rRNA sequence (Flechtner et al. 2002)! Different phenotypes (i.e. different species) within Anabaena and Aphanizomenon can be 99.9–100% similar (Lyra et al. 2001), although based on the more recent phylogenetic analysis of these two genera they will likely be combined in the future (Rajaniemi et al. 2005; Willame et al. 2006). Different species within Nodularia can be 98.7% similar (Lehktima¨ki et al. 2000). Many coccoid genera have also been found to have clear phenotypic differences that are not reflected in 16S rRNA comparisons (Palinska et al. 1996; Otsuka et al. 1998; Lyra et al. 2001). On the other hand, some cyanobacteria that are morphologically similar and simple can be quite genetically distinct (Ward et al. 1992; Casamatta et al. 2003). Because of the inadequacy of taxonomic concepts based on similarity of the 16S rRNA genes, as well as the theoretical objections to similarity-based taxonomy in general, we recommend rejection of the use of 16S rRNA dissimilarity as a primary criterion for establishment of either species or genera. Presently, there is considerable debate as to what should be the criteria for species delineation in the cyanobacteria (Johansen & Casamatta 2005). At times, morphology is insufficient to identify species with confidence. This is true for populations within the natural environment which are present in similar biotopes and which share similar but not identical morphological characteristics. The question arises as to whether the minor morphological or ecological characteristics are genetically based or simply environmentally induced. Molecular criteria for species delineation suffer from similar limitations in that strains which are

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Received 26 October 2006; accepted 22 March 2007 Associate editor: Paul Broady

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Authors Queries Journal: Phycologia Paper: phya-46-04-10 Title: Morphological and molecular characterization of selected desert soil cyanobacteria: three species new to science including Mojavia pulchra gen. et sp. nov. Dear Author During the preparation of your manuscript for publication, the questions listed below have arisen. Please attend to these matters and return this form with your proof. Many thanks for your assistance

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In the sentence beginning Stackebrandt and Goebel (1994), I rendered the author ‘‘Stackebrandt’’ in the References. Correct? Copy editor

3

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Remarks

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