The use of partial cox1, rbcL and LSU rDNA sequences for phylogenetics and species identification within the Nitzschia palea species complex (Bacillariophyceae)

Figures & data

Table 1. Isolation information for new Nitzschia strains sequenced for this study. Original strain designations correspond to the isolate names on the voucher slides held in Royal Botanic Garden of Edinburgh. See Table 2 for explanation of ‘N. palea agg.’.

Species	Strain identifier	Original strain designation	Voucher	Habitat	Locality	Isolation date
Nitzschia capitellata Hustedt	capitellata Scot1	Nit54	E4650	lake	Loch of Forfar, Forfar, UK	2008
N. capitellata	capitellata Scot2	Nit55	E4651	lake	Loch of Forfar, Forfar, UK	2008
N. cf. fonticola (Grunow) Grunow	cf. fonticola1	NIT331TM	E3963	lake	Threipmuir reservoir, near Edinburgh, UK	2003
N. cf. fonticola	cf. fonticola2	NIT328TM	E3805	lake	Threipmuir reservoir, near Edinburgh, UK	2003
N. palea agg.	New Scot1	NIT144D	E3955	lake	Dunsapie Loch, Edinburgh, UK	2008
N. palea agg.	New Scot 2	NIT335TM	E3806	lake	Threipmuir reservoir, near Edinburgh, UK	2008
N. palea agg.	New Spain1	Ter2B1	E4551	river	River Ter, near Girona, Spain	2008
N. palea agg.	New Spain2	RieraSMV	E4547	stream	Sant Martí Vell stream, near Girona, Spain	2008
N. cf. recta Grunow	cf. recta	NIT603R	E4564	pond	Royal Botanic Garden Pond, Edinburgh, UK	2007
N. sigmoidea (Nitzsch) W. Smith	sigmoidea RBGE	–	– ^a	pond	Royal Botanic Garden Pond, Edinburgh, UK	2003
^a No voucher available.

Table 2. GenBank accession numbers for the species and strains analysed. Accession numbers for sequences obtained for this study are shown in bold. Strain identifier refers to the names of the isolates as used in this paper. Within the Nitzschia palea species complex, clones are identified according to their membership of one of the three putative species (OTUs) recognized by Trobajo et al. (2009), i.e. ‘LSU clade A’, ‘mating group 1’ and ‘mating group 2’, or, for clones not belonging to any of these groups, they are referred to as ‘N. palea agg.’.

Species	Strain identifier	cox1 sequence	rbcL sequence
Cylindrotheca closterium (Ehrenberg) Reimann & Lewin	Cyl. closterium GB	AB020222	–
Cylindrotheca sp	Cylindrotheca sp GB	–	M59080
Eunotia minor (Kützing) Grunow	Eun. minor GB	–	AY571744
Navicula cf. duerrenbergiana Hustedt	Nav. cf. duerrenbergiana GB	–	AY571749
Nitzschia capitellata	Nit. capitellata Scot1	–	FN557030
N. capitellata	Nit. capitellata Scot2	–	FN557031
N. capitellata	Nit. capitellata Spain	–	FN557032
N. cf. fonticola Grunow	Nit. cf. fonticola1	FN557060	–
N. cf. fonticola	Nit. cf. fonticola2	FN557061	–
N. frustulum (Kützing) Grunow	Nit. frustulum GB	AB020225	EF423498
N. palea LSU clade A	Japan A1	FN557040	FN557018
N. palea LSU clade A	Japan B1	FN557041	FN557019
N. palea LSU clade A	Japan E	FN557043	FN557020
N. palea LSU clade A	Spain A3	FN557049	FN557022
N. palea LSU clade A	Spain A5	FN557050	FN557024
N. palea mating group 1	Belgium1	–	FN557026
N. palea mating group 1	Belgium2	–	FN557027
N. palea mating group 1	Belgium3	–	FN557028
N. palea mating group 1	Belgium4	–	FN557029
N. palea mating group 2	Paraguay	FN557048	FN557021
N. palea mating group 2	Brazil	FN557039	FN557017
N. palea mating group 2	Spain A4	–	FN557023
N. palea agg.	Egypt	FN557057	–
N. palea agg.	India	FN557056	–
N. palea agg.	Japan B2	FN557053	–
N. palea agg.	Japan C	FN557054	–
N. palea agg.	Japan D	FN557055	–
N. palea agg.	Japan F	FN557042	–
N. palea agg.	New Scot1	FN557044	–
N. palea agg.	New Scot2	FN557045	–
N. palea agg.	New Spain1	FN557046	–
N. palea agg.	New Spain2	FN557047	–
N. palea agg.	Spain B	FN557051	–
N. palea agg.	Spain C	FN557052	FN557025
N. palea agg.	SriLanka1	FN557058	–
N. palea agg.	SriLanka2	FN557063	–
N. palea agg.	UK	FN557059	–
N. cf. recta Hantzsch	Nit. cf. recta RBGE	FN557062	–
N. sigmoidea (Nitzsch) W. Smith	Nit. sigmoidea	–	FN557033
Nitzschia sp	Nit. sp AnM0026 GB	–	EU090049
Phaeodactylum tricornutum Bohlin	Pha. tricornutum GB	–	AF195952
Pseudo-nitzschia multistriata (Takano) Takano	Psn. multistriata GB	–	EF520337
Pseudo-nitzschia pungens (Grunow) Hasle	Psn. pungens GB	–	EF423507
Sellaphora pupula (Kützing) Mereschkowsky agg.	Sel. pupula1 GB	EF164937	–
S. pupula agg.	Sel. pupula2 GB	EF164958	–
Seminavis cf. robusta Danielidis & D.G. Mann	Sem. cf. robusta GB	–	AY571750

Table 3. p distances (right-top) and bp differences (left-bottom) between N. palea cox1 sequences (alignment of 495 bp). Clades with good support or identical sequences are outlined by a solid line. Shaded boxes: p distance and bp difference for the maximally different N. palea clones (UK vs New Scot2).

Fig. 1. ML tree based on cox1 gene, illustrating the phylogeny of N. palea sequences. The tree was rooted using two Sellaphora pupula sequences. Maximum parsimony, neighbour joining and maximum likelihood bootstrap values above 50% and BI posterior probabilities above 95% (MP/ NJ/ ML/ BI) are shown on the branches. Internal nodes supported by bootstrap values above 80% and posterior probabilities above 95% in at least two types of analyses are highlighted as thick lines.

Fig. 2. ML tree based on rbcL gene, illustrating the phylogeny of N. palea sequences. The tree was rooted using Eunotia minor. Maximum parsimony, neighbour joining and maximum likelihood bootstrap values above 50% and BI posterior probabilities above 95% (MP/ NJ/ ML/ BI) are shown on the branches. Internal nodes supported by bootstrap values above 80% and posterior probabilities above 95% in at least two types of analyses are highlighted as thick lines.

Table 4. p distances (right-top) and bp differences (left-bottom) between N. palea rbcL sequences (alignment of 1334 bp). Two clades with moderate to good support are outlined by solid lines, the remaining isolates, which formed a paraphyletic group, by a dashed line.

Table 5. Percentage divergence of LSU (alignment of 498 bp), cox1 (alignment of 495 bp) and rbcL (alignment of 1334 bp) among Nitzschia palea isolates. Cells filled in grey indicate intra-OTU divergences.

LSU	Clade A	Group 1	Group 2 (excl. Spain C)	Group 2 (incl. Spain C)	All others
Clade A	0.0–0.4%	–	–	–	1.0–2.4%
Group 1 (no Belgium 4)	0.6–0.8%	0.0–0.0%	–	–	0.4–1.8%
Group 2 (excl. Spain C)	1.0–1.2%	0.4–0.4%	0.0–0.4%	–	0.4–1.6%
Group 2 (incl. Spain C)	0.8–1.2%	0.4–0.4%	–	0.0–0.4%	0.2–1.6%
cox1	Clade A	Group 2 (incl. Spain C, no Spain A4)	All others
Clade A	0.0–4.6%	–	6.7–12.5%
Group 2 (incl. Spain C, no Spain A4)	9.3–11.1%		1.0–6.5%	7.9–11.1%
rbcL	Clade A	Group 1 (with Belgium 4)	Group 2 (excl. Spain C)	Group 2 (incl. Spain C)
Clade A	0.0–0.3%	–	–	–
Group 1 (with Belgium 4)	0.6–0.8%	0.0–0.0%	–	–
Group 2 (excl. Spain C)	0.7–0.9%	1.0–1.0%	0.0–0.2%	–
Group 2 (incl. Spain C)	0.5–0.9%	0.8–1.0%	–	0.0–0.6%

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