Acroprep 96 filter plates

The mtDNA control region analyses showed the presence of two major clades, a small clade separated from a large clade without any geographical pattern (Figure 1). To understand this result and determine if it is, due to the presence of nuclear copies of mitochondrial genomes or indicative of introgression, the cytochrome b gene was examined in a selection of seven samples; four from the small clade and three from the large clade (detailed methods are outlined in Supplementary Material, Text S2). A ~1000 bp fragment containing the entire cytochrome b gene was amplified using PCR primers H16064 (5’-CTTCAGTTTTTGGTTTACAAGACC-3’) and L14764 (5’-TGRTACAAAAAAATAGGMCCMGAAGG-3’) [33 (link)]. Purified PCR products (Acroprep 96 filter plates, PALL Corporation, Port Washington, NY, USA) were sequenced (BigDye Terminator v.3.1, ThermoFisher, Waltham, MA, USA) on an ABI 3730xl DNA analyser (Applied Biosystems Inc., Foster City, CA, USA) with one primer, L14764.
Sequences were aligned in Geneious^® 9.0.5 [33 (link)]. Sequences for all seven samples were aligned with a 1143 bp cytochrome b sequence of the red-billed gull (NCBI Accession No.: FM209918) and a 1143 bp cytochrome b sequence of a black-billed gull (NCBI Accession No.: FM209900.1 used as a comparison to our sequences) to determine the presence of introgression. This alignment was used to produce a neighbor-joining tree in Geneious^®.

A phenol-chloroform extraction [32 ] was used to extract genomic DNA from 100 blood samples chosen from 10 colonies across the range (Figure 1). Detailed methods for primer design and sequencing are given in Supplementary Material (Text S1). Briefly, the control region was amplified using primers BBG_CR_ND6_1F (5′-CCCCAGAACAAAACACAACC-3′) and BBG_CR_12S_1R (5′-CCCGCTCCTCTCTCCTTAGT-3′). Purified PCR products using Acroprep 96 filter plates (PALL Corporation, Port Washington, NY, USA) were sequenced with BigDye Terminator v.3.1 (ThermoFisher, Waltham, MA, USA) on an ABI 3730xl DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA). Two internal control region primers, BBG_CR_INT_R1 (5′-GCCCTGACATAGGAACCAGA-3′) and BBG_GOOSEHAIRPIN_F1 (5′-ACATCCCTCCCCAACACAT-3′), were used to sequence a 597 base pair (bp) fragment of the first domain of the control region.
Sequences were aligned with Geneious^® 9.0.5 [33 (link)]. Of the 100 extracted DNA samples, 69 samples amplified successfully. The 221 bp D-loop domain region I sequence of the red-billed gull (NCBI Accession No.: AY584133.1) and a 430 bp L. bulleri control region sequence (NCBI Accession No.: FM209657 used as a comparison to our sequences) was aligned with the black-billed gull sequences in Geneious^®. The red-billed gull sequence was used to determine how closely this species matched the black-billed gull.