DNA barcoding discriminates freshwater fishes from southeastern Nigeria and provides river system-level phylogeographic resolution within some species

Figures & data

Table I.  PCR and sequencing primers used in this study.

Name	Cocktail name/5′–3′ sequence	Taxonomic groups	References
	M13-tailed primers
	Mammal cocktail: C_VF1LFt1–C_VR1LRt1 (ratio 1:1:1:3:1:1:1:3)	Mammals, reptiles, fish	Ivanova et al. (2007)
LepF1_t1	TGTAAAACGACGGCCAGTATTCAACCAATCATAAAGATATTGG
VF1_t1	TGTAAAACGACGGCCAGTTCTCAACCAACCACAAAGACATTGG
VF1d_t1	TGTAAAACGACGGCCAGTTCTCAACCAACCACAARGAYATYGG
VF1i_t1	TGTAAAACGACGGCCAGTTCTCAACCAACCAIAAIGAIATIGG
LepR1_t1	CAGGAAACAGCTATGACTAAACTTCTGGATGTCCAAAAAATCA
VR1d_t1	CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCRAARAAYCA
VR1_t1	CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCAAAGAATCA
VR1i_t1	CAGGAAACAGCTATGACTAGACTTCTGGGTGICCIAAIAAICA
	Sequencing primers for M13-tailed PCR products
M13F	TGTAAAACGACGGCCAGT		Messing (1983)
M13R	CAGGAAACAGCTATGAC		Messing (1983)

Table II.  Summary of genetic distance (according to K2P model) for increasing taxonomic levels.

Comparison	Comparisons (n)	Minimum (%)	Mean (%)	Maximum (%)	SE
Within species	1284	0	0.17	1.08	0.01
Genus among species	1447	1.08	10.29	34.07	0.14
Family among genera	2951	0.46	17.20	24.82	0.06
Order among families	7768	15.84	21.60	28.12	0.03
Class among orders	46,609	18.46	25.05	31.96	0.01

Note: Data are from 363 sequences, 70 species, and 38 genera.

Figure 1.  NJ distance tree with 70 freshwater fish species (taxonomic families outside tree) of southeastern Nigeria, using K2P model distance calculation.

Figure 2.  Genetic variability (K2P model distance of COI sequences) within different taxonomic categories. Panel A, species; panel B, genus; panel C, family; panel D, order and panel E, class.

Table III.  Summary of southeastern Nigeria fish diversity and distribution of the genetic distance to the nearest neighbor; COI sequences of 70 species analyzed with K2P model.

Order	Family	Species (n)	< 0.1%	0.1–1.0%	1.0–2.7%	>2.7%
Siluriformes	Malapteruridae	1	0	0	0	1
	Mochokidae	4	0	0	2	2
	Clariidae	4	0	0	0	4
	Bagridae	6	0	0	2	4
	Schilbeidae	4	0	0	0	4
	Claroteidae	1	0	0	0	1
Osteoglossiformes	Gymnarchidae	1	0	0	0	1
	Notopteridae	2	0	0	0	2
	Mormyridae	10	0	0	0	10
	Arapaimidae	1	0	0	0	1
Perciformes	Cichlidae	11	0	2	3	6
	Carangidae	1	0	0	0	1
	Channidae	1	0	0	0	1
Cypriniformes	Cyprinidae	4	0	0	2	2
Lepidosireniformes	Protopteridae	2	0	0	0	2
Characiformes	Alestiidae	9	0	0	0	9
	Citharinidae	5	0	0	0	5
	Hepsetidae	1	0	0	0	1
Clupeiformes	Clupeidae	1	0	0	0	1
Gonorynchiformes	Phractolaemidae	1	0	0	0	1
	Total	70	0	2	9	59

Figure 3.  Distribution of the genetic variability (K2P model distance of COI sequences) for the 363 individuals and 70 species analyzed. Panel A, intraspecific distance and panel B, genetic distance to the nearest neighbor. SE, standard error.

Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN. 2007. Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes. 7:544–548.

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Messing J. 1983. New M13 vectors for cloning. Methods Enzymol. 101:20–78.

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