Fig. 2 Neighbor-joining tree for the COI DNA sequences of fish eggs and larvae collected from the East China Sea off the Coast of Zhoushan Archipelago. On the tree, the percentages (≥ 99%) shown next to the nodes represent the statistical supports derived from 10,000 bootstrapping permutations; each taxon is indicated with a sample code and its corresponding GenBank accession.
On the NJ tree (Fig. 2), all the sequences of 49 samples and 30 references were classified into a total of 30 smallest monophyletic clades (i.e., SMC1-30) and all sequences of the same species converged together. Each SMC had strong bootstrapping supports (value = 100%), joined by a reference sequence (ref) and sample sequences (see codes in Table 1 and Table 2). SMC1 comprised two samples (L009 and L010) and one ref of L. polyactis . SMC2 consisted of one sample (E014) and one ref of L. crocea. SMC3 consisted of two samples (E004 and E005) and one ref of C. aureus. SMC4 was composed of one sample (E027) and one ref of S. japonicus. SMC5 included two samples (E002 and E003) and one ref of A. rochei. SMC6 included three samples (E028, E029, and E030) and one ref of S. niphonicus. SMC 7, 8, 9 and 10 each had one sample (i.e. L015, L008, E026 and E001) and one ref of S. marmoratus, H. nehereus, S. macrolepis, and A. schlegelii , respectively. SMC11 had three samples (E011, E012, and E013) and one ref of K. punctatus . SMC12 had two samples (L011 and L012) and one ref of L. ocellicauda, SMC13 had one sample (L001) and one ref of A. hasta. SMC14 had three samples (E006, E007, and E008) and one ref of C. hippurus. SMC15 had one sample (L003) and one ref C. enneactis. SMC16 consisted of three sample (E020, E021, and E022) and one ref of P. argenteus. SMC17 had two samples (E010 and L006) and one ref of D. iluocoeteoides. SMC18 and SMC19 each had one sample (i.e. L007 and E019) and one ref ofE. japonicus and Minous sp ., respectively. SMC20 had two samples (E017 and E018) and one ref of Minous monodactylus.SMC21, SMC22, SMC23, SMC24 and SMC25 each included one sample (i.e. L016, E009, L004, L002, and E031) and one ref of T. xanthopterus ,Cynoglossus abbreviates , C. stigmatis, B. pterotum andS. japonica , respectively. SMC26 had three samples (E023, E024, and E025) with one ref of P. japonica. SMC27 and SMC28 each had two samples, (E032 and E033) and (L013 and L014) and one ref of T. lepturus and S. ariakensis , respectively. SMC29 had one sample (L005) and one ref of C. spinosus . SMC30 included two samples (E015 and E016) and one ref of L. japonicus. On the COI sequence data, the K-2-P distances ranged from 0.00 to 0.03 within SMCs and fell into the range of 0.15 to 0.34 between SMCs.
This study justified the identification of 30 fish species altogether from the sea, including 18 species from 33 eggs belonging into 12 families and 13 species from 16 larvae placed into 12 families. D. iluocoeteoides was identified from both the egg (E010) and the larva (L006) samples. Comparatively, DNA barcoding revealed three more egg species, C. abbreviatus (E009), M. monodactylus (E017 and E018) and M. sp. (E019), and one more larva species, L .ocellicauda (L011 and L012) than MI.