Revisiting photobiont diversity in the lichen family Verrucariaceae (Ascomycota)

Figures & data

Table 1. Amplification and sequencing primers used in this study to obtain rbcL and nuSSU sequences for Verrucariaceae photobionts. The abbreviations ‘amp’ and ‘seq’ indicate primers used for amplification and sequencing, respectively.

Marker	Target group	Primer name	Primer sequence	Direction	Use	Source
nuSSU	Heterococcus	18S SEQ 34f	GTC TCA AAG ATT AAG CCA TGC	forward	seq	Friedl (this study)
		18S SEQ 1122F	GGC TGA AAC TTA AAG GAA TTG	forward	seq	Friedl (this study)
		18S SEQ 1759R	CCT TGT TAC GAC TTC TCC TTC CTC	reverse	amp/seq	Friedl (this study)
		NS1	GTA GTC ATA TGC TTG TCT C	forward	amp	White et al. (1990)
	Trebouxiophyceae	NS1	GTA GTC ATA TGC TTG TCT C	forward	amp/seq	White et al. (1990)
		232F	CCC GAC TCG CGG TGA A	forward	amp/seq	Joneson (this study)
		NS5	AAC TTA AAG GAA TTG ACG GAA G	forward	seq	White et al. (1990)
		1006F	CCT AGT CTC AAC CAT AAA CG	forward	seq	Joneson (this study)
		18L	CAC CTA CGG AAA CCT TGT TAC GAC TT	reverse	amp/seq	Hamby et al. (1988)
		NS2	GGC TGC TGG CAC CAG ACT TGC	reverse	seq	White et al. (1990)
		NS4	CTT CCG TCA ATT CCT TTA AG	reverse	seq	White et al. (1990)
	Ulvophyceae	NS1	GTA GTC ATA TGC TTG TCT C	forward	amp	White et al. (1990)
		18L	CAC CTA CGG AAA CCT TGT TAC GAC TT	reverse	amp	Hamby et al. (1988)
		18S SEQ 1759R	CCT TGT TAC GAC TTC TCC TTC CTC	reverse	amp/seq	Friedl (this study)
		18S SEQ 20F	GTA GTC ATA TGC TTG TCT C	forward	amp/seq	Friedl (this study)
		18S SEQ 536	GAG CTG GAA TTA CCG CGG CTG CTG G	reverse	seq	Friedl (this study)
		18S SEQ 373F	TTC GAT TCC GGA GAG GGA G	forward	seq	Friedl (this study)
		18S SEQ 1122R	CAA TTC CTT TAA GTT TCA GCC	reverse	seq	Friedl (this study)
		18S SEQ 891	GTC AGA GGT GAA ATT CTT GGA	forward	seq	Friedl (this study)
		18S SEQ 422	CTA AGG GCA TCA CAG ACC TG	reverse	seq	Friedl (this study)
		18S SEQ 1122F	GGG GCT AAA TAG TAA GGG ATT G	forward	seq	Friedl (this study)
rbcL	Heterococcus	1FL	AGT GAC CGT TAT GAA TCT G	forward	amp/seq	Negrisolo et al. (2004)
		2RL	GAG AGA ACG TTT CCT TTA CT	reverse	amp/seq	Negrisolo et al. (2004)
	Trebouxiophyceae	PRASF1	ATG GTT CCA CAA ACA GAA AC	forward	amp/seq	Sherwood et al. (2000)
		PRASR1	TTG TCA ATA GTA TCA AAT TC	reverse	amp/seq	Sherwood et al. (2000)
		a-ch-rbcL-203-5′-MPN	GAA TCW TCW ACW GGW ACT TGG ACW AC	forward	amp/seq	Nelsen et al. (2011)
		a-ch-rbcL-991-3′-MPN	CCT TCT ART TTA CCW ACA AC	reverse	amp/seq	Nelsen et al. (2011)
	Ulvophyceae	PRASF1	ATG GTT CCA CAA ACA GAA AC	forward	amp/seq	Sherwood et al. (2000)
		RH1	ATG TCA CCA CAA ACA GAA ACT AAA GC	forward	amp/seq	Manhart (1994)
		DilRBCL1R	TCC ATT TGC AAG CAG CAC GGA TAA	reverse	amp/seq	Gueidan (this study)
		1385r	AAT TCA AAT TTA ATT TCT TTC C	reverse	amp/seq	Manhart (1994)

Fig. 1. Phylogenetic placement of the xanthophycean photobionts of Verrucariaceae. The tree was obtained using a maximum-likelihood analysis of a two-gene dataset (rbcL–nuSSU). Lichenized algal strains are indicated in parentheses with the name of the corresponding lichen-forming fungus and its collection number. Bootstrap values >70% are indicated above or below the branches.

Fig. 2. Phylogenetic placement of chlorophyte photobionts of Verrucariaceae. The tree was obtained using a maximum-likelihood analysis of a two-gene dataset (rbcL–nuSSU). Lichenized algal strains are indicated by the addition, in parentheses, of the name of the corresponding lichen-forming fungus. They all belong to the family Verrucariaceae, except for Cladonia grayi, Icmadophila ericetorum, Lobaria linita, Racodium rupestre, Stictis urceolatum and Xanthoria parietina. For the Verrucariaceae, the collection numbers follow the fungal names. Bootstrap values >70% are indicated above or below the branches. Photobionts with identical sequences are represented by a single taxon in the phylogeny with the names and collection numbers of each lichen indicated.

Fig. 3. Phylogenetic placement of the photobionts of Verrucariaceae related to the Prasiola-group. The tree was obtained using a maximum-likelihood analysis of a two-gene dataset (rbcL–nuSSU). Lichenized algal strains are indicated by the addition, in parentheses, of the name of the corresponding lichen-forming fungus. They all belong to the family Verrucariaceae, except for Icmadophila ericetorum, Lobaria pulmonaria, Phlyctis argena and Stictis urceolatum. For the Verrucariaceae, the collection numbers follow the fungal names. Bootstrap values >70% are indicated above or below the branches, except for values of 100%, which are indicated by a star. Photobionts with identical sequences are represented by a single taxon in the phylogeny with the names and collection numbers of each lichen indicated.

Fig. 4. Distribution of photobionts across a phylogeny of the Verrucariaceae obtained using a maximum-likelihood analysis of a four-gene dataset (RPB1–nuLSU–nuSSU–mtSSU). Lichen specimens for which molecular data are available for the photobionts are indicated in bold. For these specimens, the identity of the photobiont is shown after the taxon label using the following abbreviations: Ast = Asterochloris, Dil = Dilabifilum, Dip = Diplosphaera, Dip/dea = Diplosphaera deasonii-group (Stichococcus aff. mirabilis), Ell = Elliptochloris, Het = Heterococcus, Myr = Myrmecia, Pet = Petroderma maculiforme, Pra = Prasiola, Tre = Trebouxia. *For Hydropunctaria rheitrophila, two other specimens not included in this tree (W1048 and W0972) were associated with Heterococcus sp. (see Fig. 1).

Figs 5–11. Light photomicrographs of algae isolated from crustose lichens of the family Verrucariaceae. 5. Filamentous growth in young culture of Heterococcus sp. isolated from Verrucaria funckii and cultured on 1.5% BBM-agar (BM H. Thüs W1174). 6. Cell aggregates of Heterococcus sp. isolated from V. funckii and cultured in liquid BBM medium (BM H. Thüs W1174). 7. Dilabifilum sp. isolated from V. aquatilis (BM H. Thüs W1125). 8. Chloroidium sp. isolated from V. praetermissa (BM H. Thüs W1198). 9. Elliptochloris bilobata isolated from V. sublobulata (BM H. Thüs W0975). 10. Diplosphaera sp. isolated from V. elaeina (BM H. Thüs W1269). 11. Stichococcus cf. mirabilis isolated from Staurothele clopima (BM H. Thüs W1118). Scale bars: 10 µm.

White , TJ , Bruns , T , Lee , SB and Taylor , JW . 1990 . “ Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics ” . In A Guide to Methods and Applications, PCR Protocols , Edited by: Gelfand , M.A. , Sninsky , J.J. and White , T.J. 315 – 322 . San Diego : Academic press .

 Google Scholar

Hamby , RK , Sim , LE and Zimmer , EA . 1988 . Direct RNA sequencing: optimisation of extraction and sequencing techniques for work with higher plants . Plant. Mol. Biol. Rep. , 6 : 179 – 197 .

 Google Scholar

Negrisolo , E , Maistro , S , Incarbone , M , Moro , I , Dalla Valle , L , Broady , PA and Andreoli , C . 2004 . Morphological convergence characterizes the evolution of Xanthophyceae (Heterokontophyta): evidence from nuclear SSU rDNA and plastidial rbcL genes . Mol. Phylogenet. Evol. , 33 : 156 – 170 .

 PubMed Web of Science ®Google Scholar

Sherwood , AR , Garbary , DJ and Sheath , RG . 2000 . Assessing the phylogenetic position of the Prasiolales (Chlorophyta) using rbcL and 18S rRNA gene sequence data . Phycologia , 39 : 139 – 146 .

 Web of Science ®Google Scholar

Nelsen , MP , Rivas Plata , E , Andrew , CJ , Lücking , R and Lumbsch , HT . 2011 . Phylogenetic diversity of trentepohlialean algae associated with lichen-forming fungi . J. Phycol. , 47 : 282 – 290 .

 PubMed Web of Science ®Google Scholar

Manhart , JR . 1994 . Phylogenetic analysis of green plant rbcL sequences . Mol. Phylogenet. Evol. , 3 : 114 – 127 .

 PubMed Web of Science ®Google Scholar