Distribution and evolution of variable group-I introns in the small ribosomal subunit of North Atlantic Porphyra (Bangiales, Rhodophyta)

Figures & data

Table 1. A list of Porphyra containing the 1506 ribosomal introns sequenced for phylogenetic and intra-specific analysis.

Species	Hodgdon herbarium accession no	Date collected	Code	Collection site	GenBank accession numbers
Porphyra dioica^a	*	4/4/1998	*	North of Aberystwyth, Wales, UK	AY573850
P. leucosticta^a	67142	5/17/1997	*	Montauk Pt, New York, USA	AY573853
P. linearis^a	60868	2/18/1996	*	South Bristol, Maine, USA	AY573852
P. miniata^a	65301	6/23/1996	*	Fink Cove, Nova Scotia, Canada	AY573855
P. suborbiculata^a	*	5/20/1998	*	Masonboro, North Carolina, USA	AY573851
P. purpurea^a	65186	11/1/1996	*	Ross Island, New Brunswick, Canada	AY573854
P. umbilicalis^b	*	8/2/1995	ENG-1	North Island, Cook St. Pier, England	AY347883, AY613851^c
P. umbilicalis^b	76784	3/24/2000	ENG-2	Sidmouth, Devon, England	AY347884
P. umbilicalis^b	76783	11/8/2000	ENG-3	Overstrand, England	AY347885
P. umbilicalis^b	76578	1/21/2000	ENG-4	Sidmouth, Devon, England	AY347909
P. umbilicalis^a,b	76580	8/28/2000	IRE-1a	Clare Island, County Mayo, Ireland	AY347910
P. umbilicalis^b	76580	8/28/2000	IRE-1b	Clare Island, County Mayo, Ireland	AY347887
P. umbilicalis^b	76580	8/28/2000	IRE-2	Clare Island, County Mayo, Ireland	AY347888, AY613848^c
P. umbilicalis^b	76577	10/8/2002	NWY-1	Finnoy, NW of Alesund, Norway	AY347897
P. umbilicalis^b	76577	10/9/2002	NWY-2	Ona, NW of Alesund, Norway	AY347898
P. umbilicalis^b	*	9/21/2002	NWY-3a	Golten, Island of Sotra, Norway	AY347904
P. umbilicalis^b	*	9/21/2002	NWY-3b	Golten, Island of Sotra, Norway	AY347905
P. umbilicalis^b	76579	3/6/2001	NWY-4	Southern Norway	AY347906
P. umbilicalis^b	76579	3/6/2001	NWY-5	Southern Norway	AY347901
P. umbilicalis^b	76579	3/6/2001	NWY-6	Southern Norway	AY347908
P. umbilicalis^b	76579	3/6/2001	NWY-7	Southern Norway	AY347899, AY613850^c
P. umbilicalis^b	76579	3/6/2001	NWY-8	Southern Norway	AY347900, AY613849^c
P. umbilicalis^b	*	10/15/2002	GNY-1a	Helgoland, North Sea, Germany	AY347886
P. umbilicalis^b	74036	9/4/2000	PRT-1	Praia da Luz, Porto, Portugal	AY347907
P. umbilicalis^b	76582	7/10/2002	NBK-1	St. Martins, New Brunswick, Canada	AY347894
P. umbilicalis^b	*	7/11/2002	NBK-2	Dipper Harbor, New Brunswick, Canada	AY347895
P. umbilicalis^b	*	10/7/1996	MAE-1	Campobello Bridge, Maine, USA	AY347889
P. umbilicalis^b	72329	2/4/1996	MAE-2	Parsons Beach, Maine, USA	AY347890
P. umbilicalis^b	*	7/8/1995	MAE-3	Peaks Island, Casco Bay, Maine, USA	AY347891
P. umbilicalis^b	*	1/28/1996	MAE-4	Two Lights State Park, Maine, USA	AY347892
P. umbilicalis^b	71678	6/3/2000	MAE-5	Diamond Island, Casco Bay, Maine, USA	AY347893
P. umbilicalis^b	*	7/14/2002	NHP-1	North Wallis Sands, New Hampshire, USA	AY347896
P. umbilicalis^b	*	7/15/2002	NHP-2	Jaffrey Point, New Hampshire, USA	AY347902
P. umbilicalis^b	*	7/15/2002	NHP-3	Hampton Beach, New Hampshire, USA	AY347903
^aUsed in phylogenetic analysis; ^b Used in intra-specific analysis; ^c contains putative His–Cys open reading frame motif.

Table 2. PCR profiles used for group-I intron amplification.

Region	Primers	Fragment size	Reaction volume, µl	Taq units	MgCl2, mM	Annealing temperature, °C	Annealing time	Extension time	Cycles
1506	porint1 & porint2	Variable	50	4	2.5	56.0	1 min	1 min	35
1506	porint5 & porint6	Variable	50	1	2.25	54.3	1 min	1 min	30
1506	H501 & H502	∼471/949	50	2	1.75	56.5	1 min	1 min	36
1506 and ITS1	H50FL & UMBITSR	∼1016/1494	50	2	2.0	55.0	1 min	1 min, 30 sec	30
516	Int5pr1 & int5pr2	Variable	50	1	2.0	56.0	1 min	1 min	35

Fig. 1. An agarose gel (representing a subset of 742 Porphyra samples) illustrating an initial amplification screening for the presence of 1506 introns with flanking primers porint1 and porint2. The lanes show variable size introns or templates that lacked intron amplification. All fragments showing the 174 bp band size represent samples lacking the 1506 intron (shown as the top band of a doublet, the lower band represents primer dimer). The first lane (M) is a ΦX/Hae III DNA size marker. Numbers attached to arrows represent amplification size class and actual intron size (parentheses) in bp. Lane 1–2: P. purpurea (Orr's Island, Maine); 3–10: P. linearis (Rachel Carson Salt Pond Reserve, Bristol, Maine); 11–15, 17–19: P. umbilicalis (Peak's Island, Casco Bay, Maine); 16: Negative control; 20: P. linearis (Old Soaker, Mount Desert Island, Maine).

Table 3. Initial screening for the 1506 intron from North Atlantic Porphyra collections using flanking primers porint1 and porint 2.

	Intron Size Classes (bp)
	#Sites	N	425	555	645	725	825	1075	% w/intron
Porphyra amplissima	6	67	–	2	–	2	–	–	5.9
Porphyra dioica	2	6	–	–	–	6	–	–	100.0
Porphyra leucosticta	11	140	–	88	5	–	–	–	66.4
Porphyra linearis	12	115	–	86	–	–	8	13	93.0
Porphyra miniata	2	31	–	1	1	–	–	–	6.4
Porphyra purpurea	17	125	–	–	58	–	3	3	51.2
Porphyra suborbiculata	2	5	–	–	4	–	–	–	75.0
Porphyra umbilicalis	25	218	–	3	–	–	–	–	1.4
Porphyra yezoensis	3	10	3	3	–	–	–	–	60.0
Porphyra sp.	8	25	–	14	3	–	–	–	68.0
Total	88	742	3	197	70	8	11	16	41.2
Key:–signifies the absence of a PCR product of this size.

Table 4. The number of 1506 intron amplifications in a sub-sample (total N = 181) from the Porphyra survey. Table shows the difference in amplifications between using only flanking primers (porint 1 & porint 2) or the combination of a flanking and intron specific primer combination (porint 5 and porint 6).

	Porphyra amplissima	Porphyra dioica	Porphyra linearis	Porphyra leucosticta	Porphyra miniata	Porphyra purpurea	Porphyra suborbiculata	Porphyra umbilicalis	Porphyra yezoensis	Porphyra sp.
porint 1 + 2	3	3	37	8	0	29	1	1	4	4
porint 5 + 6	3	3	48	9	2	39	0	26	6	6
Total N	7	3	55	14	5	46	1	29	10	9

Table 5. Amplification results of 516 Group-I introns using the Int5pr1 and Int5pr2 (intron specific) primer pair from a survey of 88 Porphyra individuals.

	Porphyra amplissima	Porphyra dioica	Porphyra leucosticta	Porphyra linearis	Porphyra miniata	Porphyra purpurea	Porphyra suborbiculata	Porphyra umbilicalis	Porphyra yezoensis	Porphyra sp.
Int5pr 1 & 2 (H21)	5	3	14	10	0	10	1	9	10	2
Total N	8	3	14	15	4	21	1	9	10	3

Fig. 2. Similar putative endonuclease sequences from the Bangiophyceae within the 1506 ribosomal group-I intron. The amino acid sequence of the Porphyra umbilicalis intron ORF found in four individuals (Pumb) is aligned to the corresponding sequences from Porphyra spiralis (Psp 1), and Bangia atropurpurea (Bat 1) as given in Haugen et al. (1999). Identical positions are indicated by dots, deletions by dashes. The His-Cys box motif ( bold line) is compared to the homing endonuclease I-PpoI. Conserved residues proposed to be directly involved in zinc binding (C100, C105, H110, C125, C132, H134, C138) and the active site (H98, N119) of the I-PpoI endonuclease (Flick et al., 1998) are indicated by shading.

Fig. 3. Phylogram of the 1506 Group-I Intron from seven North Atlantic Porphyra using Maximum Parsimony analysis. Numbers represent bootstrap support from resampling of 1000 replicates. Parentheses = bootstrap support from weighted analysis.

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