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Online supporting material for Abrahamczyk, S., and S. S. Renner: The temporal build-up of hummingbird/plant mutualisms in North America and temperate South America

Table S1: The North American hummingbird species, with their ages from the chronogram Fig. S1a and their geographic distribution. Node ages are followed by 95% confidence intervals in brackets. Focal species marked in bold.


Species

Group

Age fossil calibration

Fig. S1a

Age rate calibration

Fig. S1b

Age in McGuire et al. (2014)

Distribution

Archilochus alexandri

Bee

1.5

(1.07-1.94)



1.58

(1.2-2.01)



1.01

(0.69-1.36)



North America

Archilochus colubris

Bee

1.5

(1.07-1.94)



1.58

(1.2-2.01)



1.01

(0.69-1.36)



North America

Calothorax lucifer

Bee

2.24

(1.71-2.83)



2.36

(1.83-2.86)



1.85

(1.32-2.43)



Central America to

Southern Arizona



Calypte anna

Bee

2.52

(2.01-3.13)



2.66

(2.13-3.11)



1.62

(1.23-2.03)



North America

Calypte costae

Bee

2.52

(2.01-3.13)



2.66

(2.13-3.11)



1.62

(1.23-2.03)



North America

Selasphorus platycercus

Bee

1.84

(1.41-2.26)



1.93

(1.57-2.29)



1.55

(1.05-1.61)



North America & Central America

Selasphorus rufus

Bee

0.97

(0.7-1.24)



1.02

(0.76-1.3)



1.12

(0.88-1.38)



North America

Selasphorus sasin

Bee

0.62

(0.29-0.7)



0.65

(0.45-0.86)



0.57

(0.44-0.73)



North America

Stellula calliope

Bee

0.62

(0.29-0.7)



0.65

(0.45-0.86)



0.57

(0.44-0.73)



North America

Amazilia beryllina

Emerald

-

-

0.34

(0.21-0.53)



Central America to

southern Arizona



Amazilia violiceps

Emerald

-

-

0.17 (NA)

Central America to

southern Arizona



Amazilia yucatanensis

Emerald

-

-

2.85

(2.14-3.58)



Central America to

southeastern Texas



Cynanthus latirostris

Emerald

-

-

2.87

(2.25-3.66)



Central America to

southern Arizona



Hylocharis leucotis

Emerald

-

-

2.57

(1.84-3.37)



Central America to

southern Arizona



Hylocharis xantusii

Emerald

-

-

2.57

(1.84-3.37)



Southern part of

Baja California



Eugenes fulgens

Mountain Gem

11.08

(9.5-12.8)



11.67 (10.7-12.9)

8.29

(6.92-9.56)



Central America to

southern Arizona,

New Mexico & Texas


Heliomaster constantii

Mountain Gem

-

-

5.29

(4.43-6.28)



Central America to

northern Mexico



Lampornis clemenciae

Mountain Gem

-

-

5.2

(4.39-6.21)



Central America to

southern Arizona

& Texas



Table S2: The temperate South American hummingbird species, with their ages from the chronogram Fig. S1a and their geographic distribution. Node ages are followed by 95% confidence intervals in brackets. Oreotrochilus leucopleurus is not included in our tree, and for this species we used the stem age of Oreotrochilus.


Species

Group

Age fossil calibration

Fig. S1a

Age rate calibration

Fig. S1b

Age in McGuire et al. (2014)

Distribution

Sephanoides fernandensis

Coquettes

4.62

(3.63-5.65)



4.84

(3.99-5.71)



3.13

(2.43-3.98)



Temperate South

America


Sephanoides sephaniodes

Coquettes

4.62

(3.63-5.65)



4.84

(3.99-5.71)



3.13

(2.43-3.98)



Temperate South

America


Patagona gigas

Patagoni

15.8 (NA)

17.36 (NA)

14.44 (13.05-15.77)

Andes from southern

Colombia to central Chile



Sappho sparganura

Coquettes

8.02

(6.69-9.38)



8.43

(7.47-9.43)



6.27

(5.3-7.44)



Southern Andes from

Bolivia to Argentina



Oreotrochilus (stem age)

Coquettes

8.0 (NA)

7.91 (NA)

7.78

(6.86-8.76)



Southern Andes from

Bolivia to Central Chile



Rhodopis vesper

Bees

2.23 (NA)

2.36 (NA)

1.45 (NA)

Central Andes from

Peru to Northern Chile




Table S3: Plant matrices newly clock-dated and/or used for ancestral state reconstructions for this study, 8 from North America and 8 from temperate South American; the GenBank accession numbers of a few sequences added to certain alignments (as specified below) are listed at the end.
North American clades

Aquilegia (Ranunculaceae)

Chronogram obtained by Bastida et al. (2010) under a Bayesian relaxed clock model applied to 34 accessions and 2486 aligned nucleotides of plastid and nuclear DNA sequences, using a fossil calibration. For the chronogram and ancestral state reconstruction see Figure S2a.


Castilleja (Orobanchaceae)

We used the alignment of Tank and Olmstead (2008), which comprises 79 species of Castillejinae (Orobanchaceae), representing all major lineages, two outgroup species (Paulownia tomentosa (Paulowniaceae), Rhinanthus alectorolophus (Orobanchaceae)), and 3257 aligned positions of the nuclear ribosomal DNA internal transcribed spacers ITS1 and ITS2 and the intervening 5.8S gene, the external spacer ETS and the chloroplast rps16 and trnL-F regions. A strict clock model fit the data better than a relaxed clock (ucld.stdev: 0.353), and we calibrated it using a normally distributed secondary constraint of 38.0 ± 7.5 my for the split between Paulowniaceae and Orobanchaceae, based on the age (and error range) obtained by Bell et al. (2010) for this node. With this constraint, the age inferred for the split between Rhinantheae and its sister clade was 33.53 (25.45-41.8) my. This agrees with the age inferred by Gussarova et al. (2008) for the same split, 28.3 (21.18-30.59) my, even though their tree was incorrectly rooted. For the chronogram and ancestral state reconstruction see Figure S2b.


Ipomopsis and Collomia (Polemoniaceae)

We used the alignment of Porter et al. (2010) comprising 100 taxa of Polemoniaceae (all 50 species of Ipomopsis) and 1234 aligned positions of the plastid trnL intron and trnL-trnF intergenic spacer. The nuclear ribosomal DNA internal transcribed spacers ITS1 and ITS2 and intervening 5.8S gene were omitted because of doubtfully aligned sections. A relaxed clock model fit the data better than a strict clock (ucld.mean 0.539), and we calibrated it using an average substitution rate for angiosperm plastid DNA of 1.3 substitutions/site/year x 10-9 from Richardson et al. (2001). We opted for this rate calibration instead of the Gilisenium hueberi fossil used by Porter et al. (2010) to constrain the stem of the tribe Gilieae to 98.0 my because the fossil-based dating yielded an extremely old age for Polemoniaceae compared to the age obtained for this family in the angiosperm-wide study of Bell et al. (2010). The node age inferred by us for the split between Phlox and the Ipomopsis clade, 25.84 (21.26-30.96) my, agrees with the age inferred by Bell et al. (2010) for the same node, 24.0 (12.0-37.0) my. For the chronogram see and ancestral state reconstruction Figure S2c.


Keckiella (Plantaginaceae)

We modified the alignment of Wolfe et al. (2006), which comprises all seven species of Keckiella and ten outgroup species (Plantaginaceae), 1359 aligned positions of the nuclear ribosomal DNA internal transcribed spacers ITS1 and ITS2 and the intervening 5.8S gene and the trnL-F intergenic spacer, by adding sequences of Keckiella antirrhinoides and Gambelia speciosa from GenBank. A relaxed clock model fit the data better than a strict clock (ucld.stdev ITS: 0.585, ucld.stdev trnL-F 0.822), and we calibrated it using the ITS substitution rate of 4.72 substitutions/site/year x 10-9 from Kay et al. (2005) calculated for Plantago, a closely related genus. With this calibration, the age inferred for the split between Antirrhinum and Gambelia was 5.27 (1.81-9.05) my. This agrees with the age inferred by Vargas et al. (2009) for the same node, 7.25 (10.28-4.21) my. For the chronogram and ancestral state reconstruction see Figure S2d.


Lithospermum (Boraginaceae)

Chronogram obtained by Cohen (2012) under a Bayesian relaxed clock model applied to 42 accessions and 10118 aligned nucleotides of plastid and nuclear DNA sequences, using a fossil calibration. For the chronogram and ancestral state reconstruction see Figure S2e.


Lonicera (Caprifoliaceae)

Chronogram obtained by Smith and Donoghue (2010) under a Bayesian clock model applied to 17 accessions and about 1800 aligned nucleotides of plastid and nuclear DNA sequences, using a secondary calibration. For the chronogram and ancestral state reconstruction see Figure S2f.


Monarda (Lamiaceae)

We downloaded ITS sequences from GenBank and constructed an alignment (available under www.treebase.org: No. 14591 NOT FOUND) in Mesquite (Materials and Methods), which comprised 14 out of 16 known Monarda species, five outgroup species (4 Lamiaceae and 1 Orobanchaceae), and 970 aligned positions of the nuclear ribosomal DNA internal transcribed spacers ITS1 and ITS2, together with the intervening 5.8S gene. A relaxed clock model fit the data better than a strict clock because the ucld.stdev value was 1.21, and we calibrated it with an intermediate ITS substitution rate of 1.99 substitutions/site/year x 10-9 from Kay et al. (2006) because perennial herbs, such as Monarda have been found to have medium ITS substitution rates (Soria-Hernanz et al., 2008). With this calibration, the age inferred for the split between Lamiaceae and their sister clade, containing Orobanchaceae and Paulowniaceae, was 46.76 (20.58-83.68) my. This agrees with the age inferred by Bell et al. (2010) for the same split, 48.0 (39.0-56.0) my. For the resulting chronogram and ancestral state reconstruction see Figure S2g.


Ribes (Grossulariaceae)

We used the alignment of Schultheis and Donoghue (2004), which comprises 49 of the 150 species of Ribes and two species of Itea, and has 1259 aligned positions of the nuclear ribosomal DNA external spacer (ETS), the internal transcribed spacers ITS1 and ITS2, and the intervening 5.8S gene. As an additional outgroup we added a sequence of Mitella (Saxifragaceae) from GenBank. We used a relaxed clock (ETS ucld.stdev: 1.45, ITS ucld.stdev: 0.85) and calibrated it using an ITS substitution rate of 1.2 substitutions/site/year x 10-9. This rate lies between the slow rates of trees, such as Alnus (Betulaceae; 1.1 substitutions/site/year x 10-9), and the medium rates of dwarf shrubs, such as Empetrum (Ericaceae; 1.44 substitutions/site/year x 10-9) (Kay et al., 2005). The age inferred with this rate for the split between Saxifragaceae and Iteaceae was 71.09 (35.54-111.09) my, which agrees with the age of 77.0 (60.0-88.0) my inferred by Bell et al. (2010) for the same node. The resulting chronogram and ancestral state reconstruction is shown as Figure S2h.


South American clades

Campsidium (Bignoniaceae)

We slightly modified the alignment of Olmstead et al. (2009), which comprises 86 species of Bignoniaceae, representing all major clades and most genera, two outgroup species (Verbenaceae), and 3327 aligned positions of the plastid genes rbcL and ndhF and plastid spacer trnL-trnF. A strict clock model fit the data better than a relaxed clock (relaxed clock ucld.stdev: 0.47), and we calibrated it using a plastid rate of 0.995 substitutions/site/year x 10-9 from Särkinen et al. (2007). With this calibration, the age inferred for the split between Tecomeae and its sister clade (several Bignoniaceae subfamilies) was 26.1 (22.1-30.1) my. This agrees with the age inferred by Bell et al. (2010) for the same node, 25.0 (18.0-31.0) my. For the chronogram and ancestral state reconstruction see Figure S3a.


Dendroseris (Asteraceae)

We used the plastid alignment of Kim et al. (2007), which comprises five of the eleven Dendroseris species, 60 other Sonchinae species (Asteraceae), and 580 aligned positions of the nuclear ribosomal DNA internal transcribed spacers ITS1 and ITS2 and the intervening 5.8S gene. As an additional outgroup we added a sequence of Helianthus annuus (Asteraceae) from GenBank. A relaxed clock model fit the data better than a strict clock (ucld.stdev: 1.34), and we calibrated it with an ITS substitution rate of 6.0 substitutions/site/year x 10-9 calculated by Richardson et al. (2008) for Dendroseris. With this calibration, the age inferred for the split between Cichorieae and Heliantheae was 27.33 (18.56-37.98) my. This agrees with the age of 27.0 (19.0-35.0) my inferred by Bell et al. (2010) for the same node. For the chronogram and ancestral state reconstruction see Figure S3b.


Latua (Solanaceae)

We modified the alignment of Olmstead et al. (2008), which comprises all major lineages and most genera of Solanaceae (183 species) and 3040 aligned positions of the plastid ndhF and trnL-trnF regions. A relaxed clock model fit the data better than a strict clock (ucld.stdev: 0.521), and we calibrated it with a normally distributed secondary constraint of 38.0 (29.0-47.0) my for the crown group of Solanaceae (Bell et al., 2010). MCMC chains were run for 40 million generations, sampling every 10,000th generation to reach stationarity in Tracer (Materials and Methods). The age inferred with this calibration for the Petunia clade was 9.63 (4.63-16.19) my. This agrees with the age of 12.0 my inferred for the same clade by Filipowicz and Renner (2012). For the chronogram and ancestral state reconstruction see Figure S3c.


Puya (Bromeliaceae)

We used the alignment of Jabaily and Sytsma (2010), which comprises 35 out of approximately 190 species of Puya including all basal species, 29 outgroup species (Bromeliaceae), and 2553 aligned positions of the plastid gene matK, the intergenic spacer trnS-trnG, and the rps16 intron, and the nuclear gene PHYC. A strict clock model fit the data better than a relaxed clock (ucld.stdev relaxed clock: 0.4), and because of the large plastid component of this matrix we calibrated the clock model with a plastid substitution rate of 4.87 substitutions/site/year x 10-10 from Richardson et al. (2001). With this calibration, the age inferred for the split between Puya and the Bromelioideae clade was 11.31 (9.46-13.28) my. This agrees with the age of 14.0 (7.0-22.0) my inferred by Bell et al. (2010) for the same node. For the chronogram and ancestral state reconstruction see Figure S3d.


Rhaphithamnus (Verbenaceae)

We used a reduced alignment of Marx et al. (2010), which comprises 46 species and most lineages of Verbenaceae, including both Rhaphithamnus species and 3065 aligned positions of the plastid genes ndhF, matK, rbcL, rpoC2, ccsA and rps3 and the spacer trnL-trnF. A relaxed clock model fit the data better than a strict clock (ucld.stdev: 0.774), and we calibrated it using an average plastid substitution rate of 6.0 substitutions/site/year x 10-10 calculated for the likewise woody genus Gleditsia (Fabaceae) by Schnabel and Wendel (1998). MCMC chains were run for 32 million generations, sampling every 10,000th generation to reach stationarity in Tracer. The age inferred for the split between Lantaneae and Verbeneae was 27.36 (19.12-36.87) my, which agrees with the age of 29.0 (18.0-39.0) my inferred by Bell et al. (2010) for the same node. For the chronogram and ancestral state reconstruction see Figure S3e.


Schizanthus (Solanaceae)

We modified the alignment of Pérez et al. (2006), which comprises all 12 Schizanthus species and 1566 aligned positions of the nuclear ribosomal DNA internal transcribed spacers ITS1 and ITS2, the intervening 5.8S gene, and the Waxy gene. As an additional outgroup we added GenBank sequences of Capsicum lycianthoides. A relaxed clock model fit the data better than a strict clock (ucld.stdev ITS: 0.226; Waxy 0.587), and we calibrated it with an ITS substitution rate from the biannual Rhamnaceae Phylica of 2.44 substitutions/site/year x 10-10 from Kay et al. (2006). The Waxy region was run without a rate prior. The age inferred with this calibration for the split between Schizanthus and Capsicum was 38.56 my. This agrees with the age inferred by Bell et al. (2010), 37.0 (29.0-47.0) my, for the same node. For the chronogram and ancestral state reconstruction see Figure S3f.


Tristerix (Loranthaceae)

We used the alignment of Amico et al. (2007), which comprises 11 out of 13 species of Tristerix and Ligaria, one outgroup species (Loranthaceae), and 2176 aligned positions of the nuclear ribosomal DNA internal transcribed spacers ITS1 and ITS2 and the intervening 5.8S gene, and the plastid DNA spacers trnL-trnF and atpB-rbcL. A relaxed clock model fit the data better than a strict clock (ucld.stdev: 0.994), and we calibrated it using an ITS substitution rate of 7.0 substitutions/site/year x 10-9, which is intermediate between the rates of woody Dendroseris (Asteraceae) and semi-woody Gossypium (Malvaceae). We choose this rate because Tristerix is a woody, epiphytic parasite. The plastid regions were run without a fixed rate prior. With this calibration, the age inferred for the split between Tristerix and Ligaria was 16.5 (8.23-25.68) my. This agrees with the age of 17.4 my inferred by Vidal-Russell and Nickrent (2008) for the same node. For the chronogram and ancestral state reconstruction see Figure S3g.

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