Diverse Trichomonas lineages in Australasian pigeons and doves support a columbid origin for all known Trichomonas species

Andrew Petersa, Shubhagata Dasa, Shane R. Raidala

aSchool of Animal & Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia.

Supplementary File 1

Materials and Methods

Figures S1-S4

Tables S1-S6

Supplementary References

Materials and Methods

PCR detection of Trichomonas spp.

PCR amplification of three partial gene segments (ITS1/5.8S/ITS2 rRNA, 18S rRNA and iron dehydrogenase) was performed. One of two protocols was used to amplify ITS1/5.8S/ITS2 rRNA in samples: either an approximately 326b amplicon produced by previously described primers used to genotype trichomonads (Felleisen, 1997); or an approximately 437b amplicon with newly designed primers T58SF (5’-CGCCCGTCGCTCCTACCGAT-3’) and T58SR (5’-TGCTTCAGTTCAGCGGGTCTTCC-3’). The latter protocol employed cycling conditions of 95°C for 15min, followed by 40 cycles of 94°C for 15s, 63°C for 30s and 72°C for 30s, followed by 72°C for 10min. An approximately 1.5Kb partial 18S rRNA segment was amplified using primers 16S1 and 16Sr as previously described (Cepicka et al., 2005) and an approximately 1Kb partial Fe-Fe dehydrogenase gene segment was amplified as previously described using primers TrichhydFOR and TrichhydREV (Lawson et al., 2011a). Heat-activated Taq DNA polymerase (HotStarTaq, Qiagen) with an initial denaturation step of 95°C for 15min was used for all reactions.

Phylogenetic analysis.

All discovered Trichomonas genotypes were aligned against GenBank derived sequences of other Trichomonas species and isolates, Tetratrichomonas spp. and Pentatrichomonas hominis (Tables S5, S6) using the MAFFT version 6.81b L-INS-i (for ITS1/5.8S/ITS2 rRNA and 18S rRNA genes) and FFT-NS-i x1000 (for FeHyd gene and concatenated, partitioned ITS1/5.8S/ITS2 + 18S rRNA) algorithms (Katoh et al., 2002) as implemented by Geneious. Alignments were trimmed to ensure full alignment coverage for all taxa. Aligned sequences were used to infer trees with Neighbour Joining (NJ) and Maximum Likelihood (ML:PhyML) methods (Felsenstein, 1981; Guindon et al., 2010; Saitou and Nei, 1987) using HKY85 (NJ, all genes), GTR+I+G (ML, ITS1/5.8S/ITS2 rRNA) and TIM2+I+G (ML, 18S rRNA and FeHyd) models of nucleotide substitution with 1000 bootstrap iterations in Geneious. The latter substitution models were selected as best-fit for all genes using AIC (Akaike, 1974) in jModelTest 2.1.10 (Darriba et al., 2012), which also provided invariant site proportion and gamma distribution parameters. Consensus phylograms for NJ and ML methods were produced in Geneious. Bayesian phylogenies for each gene and for concatenated ITS1/5.8S/ITS2+18S genes were inferred in BEAST 2.4.5 (Bouckaert et al., 2014). Evolutionary models for individual genes included substitution models HKY+I+G (Hasegawa et al., 1985) or GTR+I+G (Tavaré, 1986) (both with four rate categories (Yang, 1994)); strict (CLOC) (Zuckerkandl and Pauling, 1965), uncorrelated relaxed lognormal (UCLN) and uncorrelated relaxed exponential (UCED) clocks (Drummond et al., 2006); and using constant (CONST) (Kingman, 1982) and Bayesian Skyline (BSP) (Drummond et al., 2005) coalescent tree demographics. Evolutionary models for concatenated (and partitioned) genes were GTR+I+G; all combinations of strict, uncorrelated relaxed lognormal distribution and uncorrelated relaxed exponential distribution clocks for each partition (by gene); and Bayesian Skyline coalescent tree demographics. Uninformative proper priors were used including uniform [0.0,1] priors on clockRate, ucedMean and ucldMean as well as a Jeffrey’s distribution on population sizes for Bayesian Skyline coalescent models. An additional prior on the node height for all Trichomonas tips (monophyly imposed) for ITS1/5.8S/ITS2 rRNA, 18S rRNA and concatenated genes was given a normal distribution with mean 46.1, standard deviation 4.1. This prior was derived from the previous estimated time of divergence of the ptilinopine fruit-dove clade of columbid hosts, in which we predict the most basal extant

lineage of Trichomonas became ecologically isolated (Pereira et al., 2007). MCMC chain length was 50,000,000 for all BEAST runs, with parameters and trees logged every 1000 steps. Log marginal likelihoods on the twelve models of evolution for individual genes and the nine models for the concatenated genes were compared (Baele et al., 2012) with path sampling (Lartillot and Philippe, 2006) and harmonic mean estimation (Newton and Raftery, 1994) implemented with the MODEL_SELECTION package version 1.3.4 package in BEAST and in Tracer v. 1.5.0 (Rambaut et al., 2014) respectively. Path sampling had 8 steps, alpha 0.3 and chain length of 100,000 with 50% burnin and 100,000 pre-burnin. Path sampling took priority over harmonic mean estimation in model selection (Baele et al., 2012). Run output was analysed in Tracer to ensure adequate mixing and ESS on parameters. For a selected model with ESS <200 on posteriors or priors (i.e. for the ITS1/5.8S/ITS2 gene only), log and tree files from ten runs were combined with BEAST’s LogCombiner v. 2.4.5, applying 10% burnin on each run to produce a total chain length of 450,000,000. Trees were resampled to 45,000. Combined logs were loaded into Tracer and parameter statistics were analysed. All parameters were confirmed to be well-mixed and have ESS >200 (except posterior and prior on concatenated logs with 166 and 153 respectively). Trees were summarized with BEAST’s TreeAnnotator v. 2.4.5 onto the maximum clade credibility tree with a posterior probability limit of 0.0. Burnin on tree files was 10% and median node heights were applied. Output trees were visualised in FigTree 1.4.3 (Rambaut, 2016).

Ancestral state reconstruction.

The evolutionary model with highest log marginal likelihood (GTR-UCED-BSP: see phylogenetic analysis) was used for Bayesian ancestral state reconstruction on aligned ITS1/5.8S/ITS2 rRNA sequences for Trichomonas spp. Uninformative proper priors were applied as previously and discrete trait categories were applied to all taxa for geography and host. An MCMC chain length of 50,000,000 was run, generating 50,000 trees. Trees were summarized with BEAST’s TreeAnnotator v. 2.4.5 onto the maximum clade credibility tree with a posterior probability limit of 0.0. Burnin on tree files was 10% and median node heights were applied. Output trees were visualised in FigTree 1.4.3, where posterior support for ancestral biogeography (Figure 2, Table S3) and host (Figure S2) were determined.

Host divergence estimates. Mitochondrial NADH dehydrogenase subunit 2 gene sequences were acquired from GenBank for specimens (voucher where possible) of twenty species of ptilinopine (Ptilinopus-Ducula radiation) columbids, twelve species of phabine (Geophaps-Phaps radiation) columbids, Chalcophaps indica, Columba livia, Columba palumbus, Gallicolumba (Alopecoenas) beccarii, Gallicolumba luzonica, Goura scheepmakeri, Leucosarcia picata, Nesoenas mayeri, Nesoenas picturata, Otidiphaps nobilis, Streptopelia decaocto, Turtur chalcospilos, Zenaida asiatica and Zenaida macroura. Sequences were aligned in Geneious v. 10.1.3 using the MAFFT FFT-NS-i x1000 algorithm (Katoh et al., 2002). Aligned sequences were trimmed to ensure coverage over all taxa. Bayesian phylogenetic inference was performed using Beast v. 2.4.5 (see phylogenetic analysis), with MCMC chain lengths of 50,000,000 and parameter and tree logging every 1000 steps. Twelve models of evolution were explored: HKY+I+G or GTR+I+G (with four rate categories); strict, uncorrelated relaxed exponential distribution or uncorrelated lognormal distribution clocks; and constant or Bayesian Skyline demographics on the tree coalescents. Proper uninformative priors were used, including uniform [0.0,1] prior distributions on the clockRate, ucedMean and ucldMean parameters as well as a Jeffrey’s distribution on population sizes for Bayesian Skyline coalescent models. MRCA priors were added for ‘Australasian-origin’ taxa (all taxa excluding Zenaida, Columba, Nesoenas and Streptopelia), and within this clade both ptilinopine taxa and phabine taxa, with node height priors of normal distribution for each. Mean (standard deviation) node heights were 48.5 (4.2) for

Australasian-origin columbids, and 41.0 (3.8) for ptilinopines and 32.3 (3.2) for phabines, all of which were compelled into monophyly. Further MRCA priors with node height means (sd) of 46.9 (4.1) and 29.2 (3.1) respectively were provided for an ‘American’ clade of Zenaida, Columba, Nesoenas and Streptopelia and the internal clade of Old World ‘columbine’ columbids whose monophyly is supported by multigene phylogenies (Johnson et al., 2001). Tree root height of 54.4 (4.5) was also set as an MRCA prior. The height estimates (in Ma) are derived from previous multigene Bayesian inference (Pereira et al., 2007), which also provided 100% posterior support for the monophyly of these groups. All median dates (and standard deviations) were subsequently adjusted (see phylogenetic analysis above) in accordance with recent molecular estimates of dating in the Neoaves (Jarvis et al., 2014) so that the tree root height median prior became 39.4 Ma (instead of 54.4 Ma). Log marginal likelihoods were compared between models using path sampling (implemented in the package MODEL_SELECTION v. 1.3.4 in BEAST) and harmonic mean estimation (implemented in Tracer v. 1.5.0). Path sampling was performed in eight steps with an alpha of 0.3, chain length of 100,000, burnin of 50% and pre-burnin of 100,000. Path sampling took priority over harmonic mean estimation in model selection (Baele et al., 2012). Logs were analysed in Tracer to ensure adequate mixing and ESS >200 for all parameters. For the selected model (GTR+I+G, relaxed uncorrelated lognormal distribution clock and Bayesian Skyline demographics on the tree coalescent), which had ESS <200 on posteriors or priors, log and tree files from twelve runs were combined with BEAST’s LogCombiner v. 2.4.5, applying 10% burnin on each run to produce a total chain length of 540,000,000. Trees were resampled to 54,000. Combined logs were loaded into Tracer and parameter statistics were analysed. All parameters were confirmed to be well-mixed and have ESS >200 (except prior with ESS of 170). Trees were summarized with BEAST’s TreeAnnotator v. 2.4.5 onto the maximum clade credibility tree with a posterior probability limit of 0.0. Burnin on tree files was 10% and median node heights were applied. Output trees were visualised in FigTree 1.4.3 (Figure S4). For node heights used in cophylogenetic analyses, multigene dated phylogenies (Pereira et al., 2007) were used wherever possible. For those nodes without multigene dating available, median heights based on the NADH subunit 2 inference from above were used.

Host cladogram synthesis.

Host species included in the host cladogram were Alopecoenas beccarii (syn. Gallicolumba beccarii), Chalcophaps indica, Columba livia, Columba palumbus, Columbina passerina, Ducula bicolor, Geopelia cuneata, Geopelia humeralis, Geopelia striata, Geophaps scripta, Goura scheepmakeri, Hemiphaga novaeseelandiae, Leucosarcia picata, Nesoenas mayeri, Nesoenas picturata, Otidiphaps nobilis, Patagioenas fasciata, Petrophassa albipennis, Petrophassa rufipennis, Phaps chalcoptera, Ptilinopus regina, Ptilinopus ornatus, Streptopelia decaocta, Turtur chalcospilos, Zenaida asiatica and Zenaida macroura. Strongly supported (>50% ML and Bayesian) branches were included in the cladogram while other branching patterns were reduced to polytomies. Branching patterns with avian outgroups of Accipitriformes, Falconiformes, Passeriformes and Strigiformes and mammalian outgroups of Homo sapiens, Equus ferus, Canis lupus and Felis catus were created based on order-level systematics (Hackett et al., 2008; Liu et al., 2001).

Cophylogenetic analysis.

We first tested our hypothesis that the basal lineage of Trichomonas isolated in two species of fruit-doves (see phylogenetic analysis) was due to ancestral isolation of Trichomonas in the ptilinopine columbids, producing strict host-parasite codivergence. We determined the probability of homology between the branching patterns (including node heights) observed in

Trichomonas sp. 4a/b and Trichomonas sp. 5 and in their respective fruit-dove hosts, Ptilinopus regina and P. ornatus. Previous multigene phylogenies have provided strong support for these Ptilinopus species being closest relatives amongst the host taxa with known Trichomonas genotypes and we found strong support (1.0/0.97/0.99 Bayesian/ML/NJ respectively) for their associated Trichomonas genotypes (4a/4b and 5) also being closest relatives. After collapsing Trichomonas genotypes with sister taxa in single columbid hosts (i.e. 4a/4b, 3a/3b, 1a/1b, T. stableri 1/2, T. gallinae A1/A2/A3, T. gallinae B1/B2 and Trichomonas sp. H/I), a total of 23 Trichomonas taxa remained. Given an association between P. regina and Trichomonas sp. 4a/b, we calculated the probability of the nearest Trichomonas neighbour (i.e. genotype 5) being associated with P. ornatus with the formula:

P ( Host : parasite nodeassociation )= 1n−1

where n is the total number of Trichomonas genotypes (=23). Essentially we were testing our observation of the closest relative of the Trichomonas of P. regina being the Trichomonas of P. ornatus (Trichomonas sp. 5) against a random assignment of all Trichomonas genotypes to P. ornatus. The latter would be expected in a null hypothesis scenario, where no codivergent pattern was present.

We then calculated the estimated probability of the temporal proximity of given node heights for the branching of host (P. ornatus and P. regina) and associated Trichomonas genotypes (5 and 4 respectively) (see phylogenetic analysis and host divergence estimates) by comparing the observed difference between host and parasite node heights against the distribution of difference between observed host node height and a uniform distribution of zero to the maximum node height inferred for Trichomonas. Node heights were standardised to a 0-1 distribution, 1 denoting the maximum parasite node height, allowing the probability of our observations to be calculated directly from observed differences. For instance, where there is only a random association between host and parasite node height, 5% of host-parasite node height differences would be expected to be smaller than an observed host-parasite node difference of 0.025 (i.e. P=0.05). We were essentially testing our observation of branch length similarity between associated host and parasite codivergence against the difference in branch length observed under a null hypothesis scenario where parasite branch lengths are assigned randomly. Importantly, time estimates are not required and represent only relative height differences under most likely models of evolution for a given clade of host:parasite associations. Given that our hypothesis was that the Trichomonas lineage in ptilinopines became isolated with the evolution of mesocarpy in their host, we tested a range of maximum node heights for Trichomonas between 33.4 Ma and 29.7 Ma (at 0.36 Ma intervals and standardised as 0-1), which represent the earliest and latest estimated ages for the evolution of mesocarpy in Ptilinopine-Ducula radiation columbids (Pereira et al., 2007). The probability of homology between parasite and host branching pattern and depth is then summarised by:

P (homology )=P(Host : parasitenode association)× P(Host : parasite node height association)

We then tested for node height homology between all pairs of hosts and their associated parasites in the remainder of the tree. Cophylogenetic methods traditionally rely on branching homology between associated hosts and parasites to determine the probability of host-parasite codivergence but this approach makes assumptions affecting its specificity to true codivergence events and is insensitive to detecting deeply branched codivergence surrounded by numerous host-switched lineages. Assumptions essentially relate to the independence of patterns giving rise to host phylogeny from those giving rise to parasite phylogeny in a null hypothesis (i.e. no codivergence events) scenario. There are numerous real biological

situations in which these assumptions are inappropriate, typically where ecological or geographical patterns in host taxa correlate with the phylogenetic relatedness of parasites. A parasite which speciates according to the same non-host related factors may in these cases have a highly similar branching pattern to their host. An example is where vicariant speciation creates a deep pattern of phylogenetic branching in a host followed by later emergence of a parasite at the originating point of the host and subsequent spread across the vicariant barrier to infect the sister lineage. Both tree topologies would be identical, despite clearly not representing host-parasite codivergence but rather host-switching instead. We propose that improbably similar node heights in host and parasite branching provide unbiased support for host-parasite codivergence events.

We compared all host-host and associated parasite-parasite node height differences for both ITS1/5.8S/ITS2 rRNA and concatenated, partitioned ITS1/5.8S/ITS2 + 18S rRNA gene dated Bayesian Trichomonas trees (as described in phylogenetic analysis) against differences between observed host node heights and a uniform distribution of parasite node heights as described above. Methods for estimating host branching dates were as described in host divergence estimates (see also Figure S4). Four time estimates were explored for the node height of the MRCA for Trichomonas (and therefore all internal node heights, again standardised to a 0-1 distribution): 33.4 Ma (earliest estimated date for origin of mesocarpy and ecological entrapment of basal Trichomonas lineage in Ptilinopus); 29.7 Ma (latest estimated date for origin of mesocarpy corresponding to MRCA of Ptilinopine-Ducula radiation); 31.5 Ma (midpoint estimated date for origin of mesocarpy); and 62.7/72.6 Ma (alternative branching date for ITS1/5.8S/ITS2 rRNA and concatenated ITS1/5.8S/ITS2 + 18S rRNA sequences). The latter, alternative branching dates were used to test an alternative hypothesis, that the divergence between major lineages in the MRCA of all non-Ptilinopine Trichomonas taxa corresponds to the original biogeographic separation of Australasian and American columbids from a Gondwanan ancestor as hypothesised in Pereira et al (2007) but adjusted according to recent time estimates for the origin and diversification of the Neoaves (Jarvis et al., 2014). Such a hypothesis implies a date for this MRCA of 39.4 Ma, driving the MRCA of all Trichomonas taxa earlier to 62.7 or 72.6 Ma as described. For these comparisons, significant node height associations between parasite and Australasian and American host pairs were ignored (because this association was used as the calibration point). R (R Core Team, 2013) was used to carry out all cophylogenetic analyses as detailed above.

Figure S1

Bayesian inferred maximum clade credibility tree topology for concatenated and partitioned ITS1/5.8S/ITS2 + 18S rRNA genotypes of Trichomonas illustrating median node height. Evolutionary model was GTR+I+G with a relaxed uncorrelated lognormal distribution clock (ITS1/5.8S/ITS2), relaxed uncorrelated exponential distribution clock (18S) and Bayesian Skyline population demographic for tree coalescence. Isolates in bold are novel genotypes described in this study. Scale axis represented millions of years before present. Nodes are labelled alphabetically in decreasing age. Major, non-ptilinopine clades are indicated at right.

Figure S2

Bayesian reconstruction of host for common ancestors of extant Trichomonas lineages, mapped onto highest clade credibility ITS1/5.8S/ITS2 rRNA topology. Branch posterior support for the host with the highest support is provided within the genus Trichomonas. Unlabeled branches within this genus represent posterior support of 1.00. Branch color indicates discrete host category of highest support (black: columbid; red: human; blue: accipitrid; green: canid). Text color indicates most frequently identified host category for each Trichomonas genotype.

Figure S3

Cophylogram of all known Trichomonas genotypes and their associated host taxa. Bold branches and text indicate columbid or Trichomonas species. Cladogram topologies are based on multigene and NADH dehydrogenase subunit 2 gene phylogenies (host) and ITS1/5.8S/ITS2 rRNA gene consensus phylogenies (Trichomonas) (see host cladogram synthesis and cophylogenetic analysis). Associations are indicated by lines. Square boxes accompanying columbid and Trichomonas taxa indicate geographic distribution (orange: Australasia; yellow: Eurasia; blue: Americas; black: cosmopolitan). Circles indicate geographic origin (>50% support) for a host or Trichomonas clade (see ancestral state reconstruction).

Figure S4

Bayesian inferred chronogram for NADH dehydrogenase subunit 2 gene sequences of 45 columbid species, of which 23 (bold branches and text) are host to known Trichomonas genotypes (Figure S3). Median node heights (Ma) are given for branching of these Trichomonas hosts. Ptilinopine, phabine and columbine clades are indicated by green, orange and blue branch color respectively. MRCA priors for node height were provided for these clades and for the Australasian (top primary branch) and American (lower primary branch) respectively, as well as for the entire tree. Age and likely phylogenetic position of columbid fossils Primophaps schoddei29, Rupephaps taketake30 and Arenicolumba prattae31 are indicated by shaded rectangles.

Table S1.

Sites in Australasia at which columbids were sampled for Trichomonas. Species indicated with an asterisk were wild birds submitted to a wildlife hospital or carer and were not caught. 1Alopecoenas beccarii is synonymous with Gallicolumba beccarii.

No. Location Coordinates Habitat types Species caught

1 Victoria River

15°36’42”S, 131°06’56”E

Semi-arid sandstone escarpment

Petrophassa albipennis

2 Darwin 12°20’43”S, 130°53’33”E

Tropical urban woodland

Ducula bicolorGeopelia humeralisGeopelia striata

3 East Alligator River

12°26’05”S, 132°58’09”E

Tropical monsoonal savannah

Geopelia humeralisPetrophassa rufipennis

4 Cobourg Peninsula

11°09’13”S, 132°08’37”E

Tropical monsoonal littoral woodlands

Chalcophaps indicaGeopelia humeralisGeopelia striataPtilinopus regina

5 Calvert River

16°43’16”S, 137°24’57”E

Semi-arid monsoonal woodlands

Ducula bicolorPhaps chalcopteraGeopelia striata

6 Iron Range 12°35’44”S, 143°24’49”E

Tropical littoral rainforest

Ducula bicolorGeopelia humeralisPtilinopus regina

7 Low Isles 16°23’03”S, 145°33’36”E

Tropical mangrove Ducula bicolorGeopelia humeralis

8 Millaa Millaa

17°33’47”S, 145°35’25”E

Tropical rural rainforest

Macropygia amboinensis

9 Paluma Range

19°07’14”S, 146°04’45”E

Tropical woodlands Geophaps scriptaGeopelia striata

10 Nightcap Range

28°38’08”S, 153°15’39”E

Subtropical rural rainforest

Chalcophaps indica

11 Fingal Head

28°12’01”S, 153°34’09”E

Subtropical littoral rainforest

Geopelia humeralis

12 Mount Wood

29°28’57”S, 142°14’14”E

Arid vegetated stone country

Geopelia cuneata

13 Wagga Wagga

35°07’43”S, 147°19’54”E

Temperate urban garden

Ocyphaps lophotes

14 Lower Fly River

8°25’16”S, 142°55’23”E

Tropical lowland rainforest

Ducula bicolorGoura scheepmakeriGeopelia striataPtilinopus perlatus

15 Crater Mountain

6°39’21”S, 145°11’51”E

Tropical montane rainforest

Macropygia nigrirostrisOtidiphaps nobilisPtilinopus ornatusReinwardtoena reinwardtii

16 Mt Michael

6°22’49”S, 145°15’29”E

Tropical montane rainforest

Alopecoenas beccarii1

17 Batemans Bay

35°46’41”S, 150°13’33”E

Temperate forest Leucosarcia picata*

18 Currumbin 28°08’23”S, 153°29’06”E

Subtropical rainforest Ptilinopus regina*

19 Palmerston North

40°23’15”S, 175°36’56”E

Temperate rainforest Hemiphaga novaeseelandiae*

Table S2.

Representative GenBank accessions for all novel Trichomonas genotypes recovered in this study. Genes were complete ITS1/5.8S/ITS2 rRNA with partial flanking regions, partial 18S rRNA and Fe-dehydrogenase (FeHyd). Sequence data for genes marked ‘-‘ was not available. Superscript indicates culture attempted: aculture successful; bculture unsuccessful.

Trichomonas genotype(and outgroups)

Representative GenBank AccessionITS/5.8S rRNA 18S rRNA FeHyd

Trichomonas 1aa JQ027615 JQ027617 -Trichomonas 1ba JQ027616 JQ027618 -Trichomonas 1ca JX512967 JX512959 KY680343Trichomonas 1d JQ755286 JX512966 -Trichomonas 1e JQ755278 JX512965 -Trichomonas 2aa JQ030995 JQ030997 -Trichomonas 2ba JQ755288 - -Trichomonas 3a JQ692128 JQ692127 -Trichomonas 3b JQ692126 - -Trichomonas 4ab JQ755273 - -Trichomonas 4bb JQ755274 JX512962 -Trichomonas 5 JX512968 JX512960 -Trichomonas 6 JX512969 JX512961 -Trichomonas 7 KY680342 - -Trichomonas 8 KY767988 - -

Table S3.

Nodes as illustrated in Figure 2. Median age and lower and upper ages (95% highest posterior densities) are derived from Bayesian phylogenetic analysis. Support for nodes is for Bayesian, Maximum Likelihood and Neighbor Joining phylogenies respectively. Posterior probabilities from Bayesian discrete trait analysis are provided for ancestral geographic reconstruction at each node (Aus.: Australasia; Am.: Americas; Eur.: Eurasia; Afr.: Africa).

Node

Median age(Ma) Support Geography

[Aus., Am., Eur., Afr.]

Lower age(Ma)

Upper age(Ma)

A 32.9 1.0/.99/.99 0.51, 0.12, 0.31, 0.03 26.9 38.8B 19.7 .86/.46/.87 0.54, 0.09, 0.32, 0.03 6.96 31.6C 14.4 .81/-/- 0.54, 0.10, 0.32, 0.03 4.08 27.2D 10.7 1.0/.97/.99 0.84, 0.02, 0.11, 0.02 2.14 23.4E 9.49 .68/.22/.76 0.55, 0.03, 0.38, 0.03 1.66 22.5F 8.98 .99/.61/.70 0.87, 0.01, 0.11, 0.01 2.09 19.7G 7.97 .96/-/.62 0.09, 0.62, 0.22, 0.03 1.54 18.5H 6.21 .75/.53/- 0.93, 0.00, 0.05, 0.01 1.26 14.3I 3.76 .56/-/- 0.41, 0.02, 0.51, 0.05 0.429 12.8J 3.09 .99/.56/.96 0.03, 0.79, 0.13, 0.02 0.437 8.40K 2.19 .65/-/- 0.56, 0.02, 0.32, 0.10 0.180 8.40L 1.99 1.0/.94/.99 0.99, 0.00, 0.01, 0,00 0.277 5.59M 1.51 1.0/.96/.99 0.03, 0.01, 0.96, 0.01 0.109 5.43N 1.24 1.0/.66/.98 0.76, 0.01, 0.22, 0.01 0.067 5.36O 0.442 .87/.75/.87 0.81, 0.01, 0.18, 0.01 0.012 1.89P 0.412 1.0/.89/.91 0.00, 0.00, 1.0, 0.00 0.009 1.66Q 0.251 .98/.72/.89 1.0, 0.00, 0.00, 0.00 0.001 1.11R 0.169 .98/.87/.94 0.00, 0.00, 1.0, 0.00 0.000 1.19S 0.162 1.0/1.0/1.0 1.0, 0.00, 0.00, 0.00 0.000 1.33T 0.157 1.0/.92/1.0 1.0, 0.00, 0.00, 0.00 0.000 1.20U 0.141 .99/.75/.97 0.00. 1.0, 0.00, 0.00 0.000 1.14V 0.131 1.0/.85/.98 0.00. 1.0, 0.00, 0.00 0.000 0.913W 0.109 .51/.39/.71 0.00, 0.00, 1.0, 0.00 0.000 0.670

Table S4.

Host-host and associated parasite-parasite node height differences of significance (P-value <0.05) for ITS1/5.8S/ITS2 rRNA (‘single’) and concatenated, partitioned ITS/5.8S+18S rRNA Bayesian phylogenies (‘Gene’) for Trichomonas MRCA date (‘Dating’) estimates of 33.4 Ma (‘early’), 31.5 Ma (‘mid), 29.7 Ma (‘late’) and 62.7/72.6 Ma (single/concatenated trees respectively). Within-host Trichomonas divergences are not included.

Host pair Trichomonas pair P-value Gene DatingPtilinopus ornatus:Ptilinopus regina

Trichomonas sp. 5: Trichomonas sp. 4

0.023 single early

Alopecoenas beccarii:Leucosarcia picata

Trichomonas sp. 7: Trichomonas sp. 1c

0.038 single early

Phaps chalcoptera:Petrophassa albipennis

Trichomonas sp. 2a: Trichomonas sp. 1d

0.030 single early

Phaps chalcoptera:Petrophassa rufipennis

Trichomonas sp. 2a: Trichomonas sp. 1e

0.030 single early

Ptilinopus ornatus:Ptilinopus regina

Trichomonas sp. 5: Trichomonas sp. 4

0.015 single mid

Geophaps scripta:Petrophassa albipennis

Trichomonas sp. 2b: Trichomonas sp. 1d

0.016 single late

Geophaps scripta:Petrophassa rufipennis

Trichomonas sp. 2b: Trichomonas sp. 1e

0.016 single late

Table S5.

Previously reported genotypes of Trichomonas, including recorded host associations and GenBank accessions for sequences representing each genotype. Citations are 1: Felleisen (1997), 2: Grabensteiner et al. (2010), 3: Martínez-Díaz et al. (2015), 4: Sansano-Maestre et al. (2009), 5: Gerhold et al. (2008), 6: Anderson et al. (2009), 7: Kleina et al. (2004), 8: Gaspar da Silva et al. (2007), 9: Lawson et al. (2011a), 10: Lawson et al. (2011b), 11: Robinson et al. (2010), 12: Girard et al. (2014), 13: Kutisova et al. (2005), 14: Fukura et al. (1996), 15: Katiyar et al. (1995), 16: Gunderson et al. (1995), 17: Cepicka et al. (2005), 18: Cepicka et al. (2006), 19: Delgado-Viscogliosi et al. (2000).

Species Host ITS1/5.8S/ITS2 rRNA

18S rRNA Citations

T. gallinae index (A1)

Columbiformes: Columba livia

U86614 1

T. gallinae (A2) Columbiformes: Columba livia, Streptopelia decaoctaAccipitriformes: Aquila fasciata, Buteo buteo, Hieraaetus pennata

EU881912, EU881914, EU881917, FN433475, KJ776743, KM246599, KM246600

234

T. gallinae (A3) Columbiformes: Columba livia, Streptopelia decaocta

EU215362, EU215363, EU215364

EU215374, KM246607, EU215373

5

T. gallinae (B1) Columbiformes: Columba livia, Columba palumbus, Nesoenas mayeri, Nesoenas picturata, Patagioenas fasciata, Streptopelia decaocta, Zenaida macrouraAccipitriformes: Accipiter cooperii, Accipiter nisus, Aquila fasciata, Buteo buteo, Buteo lineatus, Buteo platypterus, Circus cyaneus, Milvus milvusFalconiformes: Falco tinnunculusStrigiformes: Strix aluco, Tyto albaPasseriformes: Aphelocoma californica, Carduelis carduelis, Chloris chloris, Corvus brachyrhynchos,

AY349182, EF208019, EU215369, EU290649, EU881911, EU881913, EU881915, EU881916, GQ150752, GQ150753, JQ755282, KC215387, KF993693, KF993702, KF993703, KJ776741

EU215372, EU215375

67859103114

Emberiza citrinella, Fringilla coelebs, Fringilla montifringilla, Haemorhous mexicanus, Parus major, Passer domesticus, Pica pica, Prunella modularis, Pyrrhula pyrrhula, Spinus spinus, Turdus merula

T. gallinae (B2) Columbiformes: Streptopelia decaocta,

EU215368 5

T. gypaetinii Accipitriformes: Aegypius monachus, Neophron percnopterus

KF993707, KM246601

3

Trichomonas sp. H Columbiformes: Zenaida asiatica

EU215360 5

Trichomonas sp. I Columbiformes: Zenaida asiatica

EU215361 5

Trichomonas sp. J Columbiformes: Zenaida macroura

EU215365 5

Trichomonas sp. L Columbiformes: Zenaida asiatica, Zenaida macrouraAccipitriformes: Accipiter cooperii

EU215366 EU215370 5

Trichomonas sp. GRD

Columbiformes: Columbina passerina

EU215358 EU215371 5

Trichomonas sp. GRD

Columbiformes: Columbina passerina

EU215359 5

T. stableri (1) Columbiformes: Patagioenas fasciata

EU215367, KC215389

512

T. stableri (2) Columbiformes: Patagioenas fasciata

KC215390 12

T. brixi Mammalia: Canis lupus familiaris

AY244652 AY247748 13

T. tenax Mammalia: Homo sapiens, Canis lupus familiaris, Equus ferus, Felis catus

U86615 D49495 1, 14

T. vaginalis Mammalia: Homo sapiens

L29561 U17510 15, 16

Tetratrichomonas gallinarum

Galliformes: Gallus gallus (ITS1/5.8S/ITS2 rRNA gene)Anseriformes: Anas platyrhynchos (18S)

AY244648 AY245107 13, 17

Tetratrichomonas sp. KAJ

Reptilia: Macroclemys temminckii

AY245133 AY245121 17

Tetratrichomonas Mammalia: Bison bison AY886786 18

sp. IVBPentatrichomonas hominis

Mammalia: Homo sapiens

AY245137 AF124609 17, 19

Table S6.

Previously described Trichomonas Fe-hydrogenase (FeHyd) genotypes used in this study, with host species, GenBank accession and cited reference. Superscripts refer to genotype groups of 100% identity following alignment and trimming to total coverage for all genotypes. Citations are 1: Voncken et al. (2002), 2: Lawson et al. (2011a), 3: Girard et al. (2014), 4: Chi et al. (2013), 5: McBurney et al. (2015), 6: Sansano-Maestre et al. (2016).

Species Host GenBank CitationTrichomonas gallinaea N/A AF446077 1Trichomonas vaginalis Homo sapiens AY028640 1Trichomonas gallinaeb Chloris chloris JF681136 2Trichomonas gallinaec Alectroenas pulcherrimus JF681137 2Trichomonas gallinaed Alectroenas pulcherrimus JF681138 2Trichomonas gallinaee Alectroenas pulcherrimus JF681139 2Trichomonas gallinaef Alectroenas pulcherrimus JF681140 2Trichomonas gallinaeg Nesoenas picturata JF681141 2Trichomonas gallinaeh Geopelia striata JF681142 2Trichomonas gallinaeg Patagioenas fasciata KC244200 3Trichomonas gallinaeb Patagioenas fasciata KC244201 3Trichomonas sp. Ja Zenaida macroura KC244202 3Trichomonas gallinaeb Zenaida macroura KC244203 3Trichomonas gallinaei Columba livia KC249971 3Trichomonas gallinaej Accipiter nisus KC529660 4Trichomonas gallinaek Columba palumbus KC529661 4Trichomonas gallinaei Columba palumbus KC529662 4Trichomonas gallinael Columba palumbus KC529664 4Trichomonas stableria Patagioenas fasciata KC660123 3Trichomonas stablerib Patagioenas fasciata KC660124 3Trichomonas sp. Jb Zenaida macroura KC660125 3Trichomonas sp. GRD Columbina passerina KC660126 3Trichomonas stableria Patagioenas fasciata KC660128 3Trichomonas gallinaem Columba palumbus KC962158 4Trichomonas gallinaeb Spinus tristis KJ184167 5Trichomonas gallinaen Haemorhous purpureus KJ184168 5Trichomonas gallinaeb Spinus tristis KJ184169 5Trichomonas gallinaeg Columba livia KJ184170 5Trichomonas gallinaeo Columba livia KJ184171 5Trichomonas gallinaei Columba livia KJ184172 5Trichomonas gallinaeb Accipiter gentilis KP900023 6Trichomonas gallinaeb Aquila fasciata KP900024 6Trichomonas gallinaeb Bubo bubo KP900025 6Trichomonas gallinaeb Columba livia KP900026 6Trichomonas gallinaeb Falco tinnunculus KP900027 6Trichomonas gallinaeb Strix aluco KP900028 6Trichomonas gallinaeb Tyto alba KP900029 6Trichomonas gallinaeg Columba livia KP900030 6

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