Journal of Avian Biology

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1 Journal of Avian Biology JAV Alvarez, S., salter, J. F., McCormack, J. E. and Milá, B Speciation in mountain refugia: phylogeography and demographic history of the pine and blackcapped complex. J. Avian Biol. doi: / jav Supplementary material

2 Appendix 1 Table A1. Additional information on sampling localities of pine s (S. p. pinus, S. p. macropterus and S. p. perplexus), and black-capped s (S. atriceps). Taxon Country State/Province Locality Latitude/Longitude S. p. pinus USA Alaska Juneau 58 18'08''/ '11'' S. p. pinus USA Arizona Pinaleño Mts '56''/ '29'' S. p. pinus USA California El Portal 37 45'59''/ '37'' S. p. pinus USA Montana Holter Dam 46 56'01''/ '55'' S. p. pinus USA Montana Flathead NF 47 52'00''/ '30'' S. p. pinus USA New Jersey Bear Swamp 39 17'40''/-75 05'20'' S. p. pinus USA Oregon Willamette NF 44 11'57''/ '22'' S. p. pinus USA Washington Wenatchee NF 47 01'19''/ '15'' S. p. macropterus Mexico Durango La Flor 26 34'07''/ '37'' S. p. macropterus Mexico Durango San Diego 24 56'48''/ '13'' S. p. macropterus Mexico Michoacán Rosario 19 33'50''/ '59'' S. p. perplexus Guatemala Huehuetenango Chichim 15 30'36''/-91 33'22'' S. p. perplexus Guatemala Huehuetenango Culchemal 15 31'17''/-91 28'47'' S. atriceps Guatemala Huehuetenango Chichim 15 30'36''/-91 33'22'' S. atriceps Guatemala Huehuetenango Culchemal 15 31'17''/-91 28'47'' 1

3 Figure A1. Photographs of the different taxa: Pine s S. p. pinus (A), S. p. macropterus (B), S. p. perplexus female (C) and male (D), and black-capped S. atriceps male (E). Photographs by BM. 2

4 Figure A2. Museum specimens of S. p. perplexus collected in Chiapas (region 2 in Fig. S3), Mexico, deposited at the Moore Laboratory of Zoology, shown from a ventral view (A), side view (B) and dorsal view (C). Due to DNA degradation, only small fragments of the target mtdna genes were sequenced from these specimens (see Fig. A4). A. B. C. 3

5 Figure A3. Distribution range and sampling regions of Spinus taxa in southern Mexico and Guatemala. Gray areas represent the species distribution. Region 1 extends from central Mexico through northern Mexico. Region 2 corresponds to the area around San Cristóbal de las Casas in Chiapas, Mexico, which is isolated from other highland regions. Region 3 corresponds to the highlands of Guatemala and a small region of SE Chiapas near the Tacaná volcano. The fresh tissue samples used in this study come from regions 1 and 3 (see Table 1). The few museum specimens of perplexus used in this study come from region 2, and the two macropterus specimens used as controls come from Durango and Jalisco in region 1. 4

6 Figure A4. Variable sites used to assign short sequences from 10 old museum specimens to major pine lineages. Identifiers for the old Mexican specimens are shown in red and include their respective catalog number from the Moore Laboratory of Zoology. They originate from Durango (DGO, n=1), Jalisco (JAL, n=1), and Chiapas (CHI, n=8), Mexico. Aligned sites within the gray boxes correspond to six variable sites found in a 93-bp fragment of ND2, and six variable sites found in a 41-bp fragment of ATPase 6. Hyphens represent shared nucleotides, and empty spaces represent missing data. Sites are numbered relative to the Carduelis pinus mitochondrial genome deposited in Genbank under accession KM , by Lerner et al. (2011). 5

7 Figure A5. Sampled individuals of S. p. perplexus from Huehuetenango (Guatemala). The pictures correspond to the following sample ID for females: 01N5980 (A), 01N5981 (B), 01N5994 (C), 01N6005 (D), 01N6019 (E), 01N6025 (F), and males: 01N5995 (G), 01N6017 (H), 01N6024 (I), 01N6027 (J), 01N6028 (K) and 01N6083 (L). Photographs for male number 01N6012 not available. Photographs by BM. 6

8 Appendix 2. List of the 97 specimens used in the study, including specimen ID, species code (BCSI = black-capped, BHSI = black-headed, PISI = pine ), locality information, and haplotype designation for target molecular markers. Asterisks refer to the shorter fragments of mtdna genes amplified for old museum specimens from the Moore Laboratory of Zoology (see Fig. A4). For the Genbank accession of each haplotype see Appendix 3. Specimen ID Species State Country ND2 ATP BRM15 GAPDH AldoB 03N1055 BCSI 24 (Y2) 22 (Y1) 4 1, N1056 BCSI 23 (Y1) 23 (Y2) N6034 BCSI 24 (Y2) 22 (Y1) 4 1, N6050 BCSI 23 (Y1) 24 (Y3) 4 2, N1083 BHSI X2 X2 6, 7 7 2, 3 01N7017 BHSI Peten Guatemala X1 X1 5 5, N5994 PISI 21 (A1) 20 (A1) N5995 PISI 21 (A1) 20 (A1) N6005 PISI 21 (A1) 20 (A1) N6012 PISI 22 (A2) 20 (A1) 1, 3 1 1, 5 01N6017 PISI 21 (A1) 21 (A2) 1, 3 1 1, 5 01N6024 PISI 21 (A1) 01N6027 PISI 21 (A1) 20 (A1) 1, N6028 PISI 21 (A1) 21 (A2) 1, N5980 PISI 22 (A2) 20 (A1) 3 1, N5981 PISI 21 (A1) 21 (A2) N6025 PISI 21 (A1) 20 (A1) N6019 PISI 21 (A1) 01N6083 PISI 22 (A2) 20 (A1) 1, N5701 PISI Durango Mexico 1 (B) 4 (E) N5702 PISI Durango Mexico 1 (B) 1 (B) N5703 PISI Durango Mexico 18 (S) 1 (B) N5704 PISI Durango Mexico 1 (B) 1 (B) N5705 PISI Durango Mexico 1 (B) 1 (B) N5706 PISI Durango Mexico 1 (B) 1 (B) 1 1 1, 6 01N5707 PISI Durango Mexico 6 (G) 1 (B) N5708 PISI Durango Mexico 1 (B) 1 (B) N5712 PISI Durango Mexico 1 (B) 5 (F) N5718 PISI Durango Mexico 1 (B) 1 (B) N5721 PISI Durango Mexico 15 (P) 01N5722 PISI Durango Mexico 1 (B) 01N5723 PISI Durango Mexico 1 (B) 01N5728 PISI Durango Mexico 14 (O) 01N5732 PISI Durango Mexico 1 (B) 01N5743 PISI Durango Mexico 1 (B) 1 (B) 01N5744 PISI Durango Mexico 5 (F) 1 (B) 01N5745 PISI Durango Mexico 10 (K) 01N5746 PISI Durango Mexico 1 (B) 6 (G) 7

9 01N5747 PISI Durango Mexico 1 (B) 1 (B) 01N5748 PISI Durango Mexico 13 (N) 01N5750 PISI Durango Mexico 16 (Q) 7 (H) 01N5751 PISI Durango Mexico 8 (I) 8 (I) 01N5752 PISI Durango Mexico 1 (B) 9 (J) 01N5754 PISI Durango Mexico 19 (T) 10 (K) 01N5755 PISI Durango Mexico 6 (G) 1 (B) 01N5756 PISI Durango Mexico 1 (B) 11 (L) 01N5757 PISI Durango Mexico 1 (B) 1 (B) 99N0026 PISI Michoacan Mexico 1 (B) 1 (B) 99N0071 PISI Michoacan Mexico 1 (B) 2 (C ) 99N0095 PISI Michoacan Mexico 1 (B) 99N0096 PISI Michoacan Mexico 4 3 (D) 99N0101 PISI Michoacan Mexico 1 (B) 1 (B) 02N7319 PISI Alaska USA 13 (N) 1 (B) 02N7320 PISI Alaska USA 1 (B) 02N7321 PISI Alaska USA 1 (B) 1 (B) 02N7322 PISI Alaska USA 1 (B) 1 (B) 02N7323 PISI Alaska USA 6 (G) 1 (B) 00N6678 PISI California USA 1 (B) 1 (B) N6679 PISI California USA 19 (T) 16 (Q) N6680 PISI California USA 1 (B) 1 (B) N6681 PISI California USA 3 (D) N6682 PISI California USA 15 (P) 1 (B) N6683 PISI California USA 6 (G) 1 (B) 00N6685 PISI California USA 12 (M) 1 (B) 00N6687 PISI California USA 16 (Q) 17 (R ) 00N6694 PISI California USA 1 (B) 00N6695 PISI California USA 6 (G) 1 (B) 00N5185 PISI Montana USA 1 (B) 00N5189 PISI Montana USA 13 (N) 15 (P) N7776 PISI Montana USA 1 (B) 18 (T) N7779 PISI Montana USA 16 (Q) 17 (R ) N7793 PISI Montana USA 1 (B) 6 (G) N7798 PISI Montana USA 5 (F) 1 (B) N9446 PISI Montana USA 2 ( C) 15 (P) 01N9447 PISI Montana USA 6 (G) 01N9448 PISI Montana USA 19 (T) 19 01N7500 PISI New Jersey USA 1 (B) 1 (B) 01N7501 PISI New Jersey USA 7 (H) 1 (B) 01N7502 PISI New Jersey USA 20 (V) 1 (B) 99N7290 PISI Oregon USA 17 (R ) 1 (B) 99N7762 PISI Oregon USA 11 (L) 13 99N7763 PISI Oregon USA 1 (B) 14 99N7764 PISI Oregon USA 1 (B) 1 (B) 8

10 99N7196 PISI Washington USA 1 (B) 12 99N7200 PISI Washington USA 9 (J) 1 (B) 99N7202 PISI Washington USA 1 (B) 1 (B) MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Chiapas Mexico * * MLZ PISI Durango Mexico * * MLZ PISI Jalisco Mexico * * 9

11 Appendix 3. Genbank accessions and associated specimens. Accession Locus Haplotype Sample ID Species State Country KT ND2 B (1) 02N7322 Pine Alaska USA KT ND3 C (2) 01N9446 Pine Montana USA KT ND4 D (3) 00N6681 Pine California USA KT ND5 E (4) 99N0096 Pine Michoacan Mexico KT ND6 F (5) 01N5744 Pine Durango Mexico KT ND7 G (6) 00N6683 Pine California USA KT ND8 H (7) 01N7501 Pine New Jersey USA KT ND9 I (8) 01N5751 Pine Durango Mexico KT ND10 J (9) 99N7200 Pine Washington USA KT ND11 K (10) 01N5745 Pine Durango Mexico KT ND12 L (11) 99N7762 Pine Oregon USA KT ND13 M (12) 00N6685 Pine California USA KT ND14 N (13) 00N5189 Pine Montana USA KT ND15 O (14) 01N5728 Pine Durango Mexico KT ND16 P (15) 00N6682 Pine California USA KT ND17 Q (16) 00N6687 Pine California USA KT ND18 R (17) 99N7290 Pine Oregon USA KT ND19 S (18) 01N5703 Pine Durango Mexico KT ND20 T (19) 01N9448 Pine Montana USA KT ND21 V (20) 01N7502 Pine New Jersey USA KT ND22 A1 (21) 01N5981 Pine KT ND23 A2 (22) 01N6012 Pine KT ND24 Y1 (23) 03N1056 KT ND25 Y2 (24) 01N6034 KT ND26 X1 01N7017 KT ND27 X2 03N1083 Peten Guatemala KT ATPase8&6 B (1) 99N0026 Pine Michoacan Mexico KT ATPase8&6 C (2) 99N0071 Pine Michoacan Mexico KT ATPase8&6 D (3) 99N0096 Pine Michoacan Mexico KT ATPase8&6 E (4) 01N5701 Pine Durango Mexico 10

12 KT ATPase8&6 F (5) 01N5712 Pine Durango Mexico KT ATPase8&6 G (6) 01N5746 Pine Durango Mexico KT ATPase8&6 H (7) 01N5750 Pine Durango Mexico KT ATPase8&6 I (8) 01N5751 Pine Durango Mexico KT ATPase8&6 J (9) 01N5752 Pine Durango Mexico KT ATPase8&6 K (10) 01N5754 Pine Durango Mexico KT ATPase8&6 L (11) 01N5756 Pine Durango Mexico KT ATPase8&6 M (12) 99N7196 Pine Washington USA KT ATPase8&6 N (13) 99N7762 Pine Oregon USA KT ATPase8&6 O (14) 99N7763 Pine Oregon USA KT ATPase8&6 P (15) 00N5189 Pine Montana USA KT ATPase8&6 Q (16) 00N6679 Pine California USA KT ATPase8&6 R (17) 00N6687 Pine California USA KT ATPase8&6 T (18) 01N7776 Pine Montana USA KT ATPase8&6 U (19) 01N9448 Pine Montana USA KT ATPase8&6 A1 (20) 01N5980 Pine KT ATPase8&6 A2 (21) 01N5981 Pine KT ATPase8&6 Y1 (22) 03N1055 KT ATPase8&6 Y2 (23) 03N1056 KT ATPase8&6 Y3 (24) 01N6050 KT ATPase8&6 X1 01N7017 KT ATPase8&6 X2 03N1083 Peten Guatemala KT BRM 1 01N5702 Pine Durango Mexico KT BRM 2 01N5701 Pine Durango Mexico KT BRM 3 01N5995 Pine KT BRM 4 01N6050 KT BRM 5 01N7017 KT BRM 6 03N1083 KT BRM 7 03N1083 Peten Guatemala 11

13 KT GAPDH 1 01N5702 Pine Durango Mexico KT GAPDH 1 01N6034 KT GAPDH 2 03N1056 KT GAPDH 3 01N6050 KT GAPDH 4 01N5980 Pine KT GAPDH 5 01N7017 KT GAPDH 6 01N7017 KT GAPDH 7 03N1083 Peten Guatemala Peten Guatemala KT AldoB 1 01N5702 Pine Durango Mexico KT AldoB 2 03N1083 KT AldoB 3 03N1083 KT AldoB 4 01N7017 Peten Guatemala KT AldoB 5 01N6012 Pine KT AldoB 5 03N1056 KT AldoB 6 01N5706 Pine Durango Mexico 12

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