Genetic differentiation and distribution of Pyropia acanthophora (Bangiales, Rhodophyta) in the Philippines

Figures & data

Table 1: Summary of the currently known introduced Pyropia species.

Species	Probable vector of introduction	Origin	Extent (Reference)
Py. acanthophora (E. C. Oliveira & Coll) M. C. Oliveira, D. Milstein & E. C. Oliveira	ballast water, shipping, aquaculture	Indo-Pacific	Brazil (Milstein et al., 2015)
Py. katadae (A. Miura) M.S. Hwang, H.G. Choi, N. Kikuchi & M. Miyata	shipping	Northwestern Pacific: probably Japan or China	Atlantic United States: New England (Neefus et al., 2008)
Py. leucosticta (Thuret) C.D. Neefus & J. Brodie	shipping	Pacific	North Atlantic Ocean: Great Britain and Ireland (Brodie et al., 1998)
Py. koreana (M. S. Hwang & I. K. Lee) M. S. Hwang, H. G. Choi, Y. S. Oh & I. K. Lee	no data	Northwestern Pacific	Balearic Is., Spain (Vergés et al., 2013b as Py. olivii), New Zealand (Nelson et al., 2014)
Py. suborbiculata (Kjellman) J.E. Sutherland, H.G. Choi, M.S. Hwang & W.A. Nelson	shipping	Northwestern Pacific: Japan, China	Atlantic United States: Connecticut, Delaware, North Carolina, Canary Islands, Mexico, New Zealand (Humm, 1979 as Po. carolinensis J. Coll & J. Cox; Broom et al., 2002; Aguilar-Rosas & Aguilar-Rosas, 2003; Neefus et al., 2008), Brazil (Torrano-Silva et al., 2010; Milstein et al., 2015), and Europe: Northeastern Atlantic & Mediterranean coasts (Haroun et al., 2002, as Po. carolinensis; Milstein et al., 2012; Vergés et al., 2013a; Sànchez et al., 2014)
Py. tanegashimensis (Shinmura) N. Kikuchi & E. Fijiyoshi	ballast water, shipping, aquaculture	Indo-Pacific	Brazil (Milstein et al. 2015)
Py. vietnamensis (Tak. Tanaka & Pham-Hoàng Ho) J.E. Sutherland & Monotilla	ballast water, shipping, aquaculture	Indo-Pacific	Brazil (Milstein et al. 2015)
Py. yezoensis (Ueda) M.S. Hwang & H. G. Choi (f. yezoensis & f. narawaensis)	shellfish aquaculture, shipping	North Asia and/or East Asia Pacific: Japan	Mediterranean Sea: Thau Lagoon (Verlaque, 2001), Northeastern America (Yarish et al., 1998): Maine (Watson et al., 1998, 1999), New Hampshire (West et al., 2005), New England (Neefus et al., 2008)

Table 2. Summary of sampling locations and haplotype information of Philippine Py. acanthophora specimens used in this study.

Site Abbreviation	Collecting site (Coordinates)	Isolate	Haplotype		Genbank Accession Number
			rbcL	COI-5P	rbcL	COI-5P
APA1	Brgy. San Antonio, Aparri, Cagayan (18°35’97” N, 121°64’12” E)	APA1.1	R5	C11	KU234722	KX036865
		APA1.2	R6	C11	KU234723	KX036866
		APA1.3	R10	C11	KU234724	KX036867
		APA1.4	R7	C11	KU234725	KX036868
		APA1.5	R20	C8	KU234726	KX036869
		APA1.6	R10	C11	KU234727	KX036870
APA2	Brgy. San Antonio-Punta Boundary, Aparri, Cagayan (18°36’08” N, 121°63’65” E)	APA2.1	R10	C11	KU234728	KX036871
		APA2.2	R10	C11	KU234729	KX036872
		APA2.3	R10	C11	KU234730	KX036873
APA3	Brgy. Punta (Port Aparri,) Aparri, Cagayan (18°36’29’’ N, 121°62’86” E)	APA3.1	R22	C11	KU234731	KX036874
		APA3.2	R10	C11	KU234732	KX036875
		APA3.3	R10	C11	KU234733	KX036876
BUR	Brgy. Saoit, Burgos, Ilocos Norte (18°53’82” N, 120°65’74’’ E)	BUR1	R11	C8	KU234734	KX036877
		BUR2	R11	C8	KU234735	KX036878
		BUR3	R11	C8	KU234736	KX036879
CAM	Sitio Minabel, Camiguin Is., Cagayan (19°00’23” N, 121°94’48” E)	CAM1	R8	C8	KU234737	KX036880
		CAM2	R5	C8	KU234738	KX036881
		CAM3	R5	C8	KU234739	KX036882
		CAM4	R5	C8	KU234740	KX036883
		CAM5	R5	C8	KU234741	KX036884
		CAM6	R5	C8	KU234742	KX036885
DLE	De Leaño, Claveria, Cagayan (18°62’16” N, 121°09’34” E)	DLE1	R9	C4	KU234743	KX036886
		DLE2	R9	C4	KU234744	KX036887
MIN	Mingay, Brgy. San Juan, Sta. Praxedes, Cagayan (18°59’86” N, 120°99’09” E)	MIN1	R21	C12	KU234745	KX036888
		MIN2	R19	C4	KU234746	KX036889
		MIN3	R19	C4	KU234747	KX036890
NAN	Sitio Nanaplaan, Brgy. San Juan, Sta. Praxedes, Cagayan (18°59’92” N, 120°99’00” E)	NAN1	R19	C4	KU234748	KX036891
		NAN2	R19	C4	KU234749	KX036892
		NAN3	R19	C4	KU234750	KX036893
PAM	Pamoctan Is., Camiguin, Cagayan (18°89’98” N, 121°83’82” E)	PAM1	R16	C8	KU234751	KX036894
		PAM2	R16	C3	KU234752	KX036895
		PAM3	R16	C8	KU234753	KX036896
		PAM4	R16	C8	KU234754	KX036897
		PAM5	R17	C8	KU234755	KX036898
		PAM6	R17	C8	KU234756	KX036899
		PAM7	R16	C8	KU234757	KX036900
		PAM8	R16	C8	KU234758	KX036901
		PAM9	R18	C8	KU234759	KX036902
PAG	Pagudpud, Ilocos Norte (18°61’46” N, 120°86’96’’ E)	PAG1	R11	C8	KU234760	KX036903
		PAG2	R11	C8	KU234761	KX036904
		PAG3	R11	C8	KU234762	KX036905
PAT	Sitio Malolog, Patunungan, Sta. Ana, Cagayan (18°44’41” N, 122°25’92” E)	PAT1	R14	C8	KU234763	KX036906
		PAT2	R14	C8	KU234764	KX036907
		PAT3	R15	C8	KU234765	KX036908
PEA	Pata East, Claveria, Cagayan (18°62’52” N, 121°09’66” E)	PEA1	R13	C8	KU234766	KX036909
		PEA2	R13	C8	KU234767	KX036910
		PEA3	R23	C8	KU234768	KX036911
TAG	Taggat, Claveria, Cagayan (18°61’35” N, 121°04’79” E)	TAG1	R12	C4	KU234769	KX036912
		TAG2	R12	C4	KU234770	KX036913
		TAG3	R5	C4	KU234771	KX036914

Fig. 1. Phylogenetic tree based on Bayesian Inference (BI) analysis using COI-5P gene sequences. C1-12 represents the haplotype clades. The numbers at the nodes indicate posterior probabilities support values.

Fig. 2. Phylogenetic tree based on Bayesian Inference (BI) analysis using rbcL gene sequences. R1-20 represents the haplotype clades. The numbers at the nodes indicate posterior probabilities support values.

Figs 3–4. Statistical parsimony analysis representing the entire Py. acanthophora haplotypes inferred from Fig. 3 COI-5P and Fig. 4 rbcL using TCS. The size of the circles represents the number of individuals; black dots correspond to missing haplotypes.

Figs 5–6. Statistical parsimony representing the entire Py. acanthophora haplotypes inferred from the combined COI-5P-rbcL dataset using TCS and their geographic distribution. Fig. 5. Haplotype network for COI-5P-rbcL dataset for all the known native populations of Py. acanthophora. The size of the circles represents the number of individuals; black dots correspond to missing haplotypes. Fig. 6. The geographic distribution of native haplotypes of Py. acanthophora in Hong Kong, Taiwan, and India. In the Philippines, the native populations of Py. acanthophora are limited across Northern Philippines (boxed).

Fig. 7. The geographic distribution of the combined COI-5P-rbcL haplotypes of Py. acanthophora within the Northern Philippines.

Table 3. Genetic diversity statistics of Py. acanthophora for each geographic location calculated for mitochondrial COI-5P and plastid rbcL sequences.

Locus	Location	N	Nh	k	h	π (x 10-2)	Tajima’s D	Fu & Li’s D*
COI-5P (mtDNA)	Philippines	50	5	0.820	0.632 ± 0.051	0.135 ± 0.019	−0.64231	−1.79190
	Taiwan	19	8	1.111	0.766 ± 0.092	0.183 ± 0.038	−1.766001	−1.99546
	Hong Kong	1	1	-	-	-	-	-
	India	4	1	-	-	-	-	-
	Hawaii, USA	2	1	-	-	-	-	-
	Brazil	8	2	0.429	0.429 ± 0.169	0.071± 0.028	0.33350	0.088779
	Total	84	12	0.910	0.675 ± 0.049	0.150 ± 0.017	−1.68927	−1.55984
rbcL (cpDNA)	Philippines	50	19	4.243	0.929 ± 0.016	0.322 ± 0.023	0.00108	-0.25860
	Taiwan	13	2	0.385	0.385 ± 0.132	0.029 ± 0.010	0.4256	0.73235
	Japan	2	1	-	-	-	-	-
	Hong Kong	1	1	-	-	-	-	-
	India	1	1	-	-	-	-	-
	Brazil	1	1	-	-	-	-	-
	Total	68	23	4.655	0.935 ± 0.012	0.353 ± 0.021	−0.09542	−0.77453

N: number of individuals, Nh: number of haplotypes, k: average sequence divergence, h: haplotype diversity (standard deviation), π: nucleotide diversity (standard deviation)

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