Nestling S Mantle Feathers from 51 Egretta Eulophotes, 24 Egretta Garzetta, 20 Bubulcus

Sampling

Nestling’s mantle feathers from 51 Egretta eulophotes, 24 Egretta garzetta, 20 Bubulcus ibis and 39 Ardeola bacchus were plucked during 2002-2009 and their species identities were based on the morphological identification of their parents; while flight feather samples of 3

Ardea alba and 2 Mesophoyx intermedia were opportunistically collected from dead adults during fieldwork (a total of 139 individual, Table S1). We also collected 88 white naturally molted feathers from three heronries at the breeding season of 2009 (Table S1). The molted feathers were large (more than 8 cm long, mostly flight feathers or tail feathers) and of white color, which distinguished them from feathers of other non-ardeid species. The plucked feather samples were kept individually in 95% ethanol at -20 °C for subsequent analysis, while the molted feather samples were placed individually in paper envelopes and stored at 4

°C until processing .

Table S1 Names and locations of Ardeids’ nesting colonies accessed, nesting species and numbers of molted and plucked feathers collected

Plucked feathers Molted feathers Species Location Coordinates N Species Location Coordinates N Egretta eulophotes Zhangzhou 23°47’12"N, 117°43’39"E 20 Fuding 27° 1'29"N, 120°25'7"E 31 Xiamen 24°26’6"N, 31 Egretta garzetta Yancheng 33° 1'54"N, 120°49'35"E 6 118°0’19"E Fuzhou 26°12’7"N, 119°34’37"E 1 Ardeid sp. Zhangzhou 23°47’12"N, 35 Quanzhou 26°9’35"N, 118°11’1"E 1 117°43’39"E Xiamen 24°26’6"N, 118°0’19"E 8 Haikou 19°47’4"N, 22 Shantou 23°17'20"N, 116°44'46"E 8 110°0’27"E Ardea alba Fuzhou 26°12’7"N, 119°34’37"E 1 Quanzhou 26°9’35"N, 118°11’1"E 2 Bubulcus ibis Yancheng 33° 1'54"N, 120°49'35"E 7 Xiamen 24°26’6"N, 118°0’19"E 1 Haikou 19°29’21"N, 109°27’57"E 12 Mesophoyx Xiamen 24°26’50"N, 118°3’27"E 2 intermedia

1 Ardeola bacchus Fuzhou 26°12’7"N, 119°34’37"E 6 Quanzhou 26°9’35"N, 118°11’1"E 2 Xiamen 24°26’6"N, 118°0’19"E 2 Haikou 19°29’21"N, 109°27’57"E 29 Total 139 88

DNA isolation

Genomic DNA was isolated from the Ardeid feathers and a negative control containing no

Ardeid tissue was included in each group of DNA extractions. The operational procedure was as follows:

(1) Samples were cut into small pieces (c. 5 mm) and incubated overnight at 55°C in 600 µL of lysis buffer (50 mM Tris-Cl pH 8.0, 50 mM EDTA, 0.01 mg Protease K and 1% sodium dodecyl sulphate).

(2) Add 200 µL of 7.5 M ammonium acetate for protein precipitation , and centrifuge at 5,000 g for 3 min at 4°C.

(3) Transfer the supernatant to a fresh microfuge tube, add isopyknic phenol-chloroform- isoamylol (25:24:1) and mix the organic and aqueous phases for 1 minute. Then, centrifuge at

10,000 g for 10 min at 15°C.

(4) Transfer the supernatant to a fresh microfuge tube, and repeat step (3) once.

(5) Transfer the upper aqueous phase to a fresh microfuge tube, collect the DNA by standard precipitation with isopyknic isopropanol for 30 minutes at 0°C, and recover the precipitate of nucleic acids by centrifugation at 10,000 g for 5 minutes at 4°C.

(6) Remove the supernatant by aspiration, rinse the pellet with 500 μl of 75% ethanol in H2O, and centrifuge the tubes at 10,000 g for 5 minute at 4°C.

2 (7) Repeat step (6) once.

(8) Remove the supernatant by aspiration, dry the pellet in the air, and redissolve the pellet in

30 μl of TE (pH 7.6).

References Mino CI, Del Lama SN (2009) Molted Feathers as a Source of DNA for Genetic Studies in Waterbird

Populations. Waterbirds 32: 322-329

Rudnick JA, Katzner TE, Bragin EA, Rhodes OE, Dewoody JA (2005) Using naturally shed feathers for

individual identification, genetic parentage analyses, and population monitoring in an endangered

Eastern imperial eagle (Aquila heliaca) population from Kazakhstan. Mol Ecol 14: 2959-2967

Segelbacher G (2002) Noninvasive genetic analysis in birds: testing reliability of feather samples. Mol Ecol

Notes 2: 367-369