ILLINOIS NATURAL HISTORY SURVEY BULLETIN 1 A molecular phylogenetic analysis of the genera of fruit doves and their allies using dense taxonomic sampling Jennifer E. Nowak1†, Andrew D. Sweet1,2†, Jason D. Weckstein3,4, and Kevin P. Johnson1 1 Illinois Natural History Survey, Prairie Research Institute, University of Illinois at Urbana-Champaign, 1816 South Oak Street, Champaign, Illinois 61820, USA 2 Department of Entomology, Purdue University, 901 West State Street, West Lafayette, Indiana 47907, USA 3 Ornithology Department, Academy of Natural Sciences, 1900 Benjamin Franklin Parkway, Philadelphia, Pennsylvania 19103, USA 4 Department of Biodiversity, Earth, and Environmental Sciences, Drexel University, 1900 Benjamin Franklin Parkway, Philadelphia, Pennsylvania 19103, USA † Authors contributed equally to the work Research Article Cite This Article: Nowak, J. E., A. D. Sweet, J. D. Weckstein, and K. P. Johnson. 2019. A molecular phylogenetic analysis of the genera of fruit doves and their allies using dense taxonomic sampling. Illinois Natural History Survey Bulletin 42:2019001. Author For Correspondence: Andrew D. Sweet email: sweet19@purdue.edu Data Accessibility Statement Sequence data are available on GenBank under accession numbers KT023313-KT023498 and KT029857-KT029914. Relevant sequence alignment and phylo- genetic tree files are available from the Illinois Data Bank (DOI: 10.13012/B2IDB-9797270_V1). Keywords: Raphinae, phylogeny, foraging, dispersal, biogeography, taxon sampling Received: 23 May 2019 Accepted: 16 July 2019 Associate Editor: Auriel M. V. Fournier Editor in Chief: Maximilian L. Allen Abstract Fruit doves and their allies are a diverse group within the pigeon and dove family (Aves: Columbidae). Progress toward subfamilial classification of Columbidae relies on identifying major groups and the phylogenetic relationships within these groups. One such recently proposed group is the Raphinae, based on previous evidence that the extinct dodo is potentially within what was formerly recognized as the Treroninae (fruit doves and allies). Although several studies have explored the phylogenetic relationships within Columbidae, most have focused either on broad-scale, familial-level relationships or finer-scale, species-level relationships. Here we use mitochondrial and nuclear gene sequences from a diverse taxonomic sample to identify relationships among the genera and species of fruit doves and their allies. In particular, our goal is to identify which of these genera should be included within Raphinae (the name that has taxonomic priority over Treroninae), focusing on an inclusive, well-supported, monophyletic group. We also use dense taxon sampling to explore relationships among genera and species in this group, expanding on previous studies. In addition, we use resulting phylogenetic hypotheses to recon- struct the ancestral evolutionary history of foraging mode and biogeographic patterns of dispersal within the group. We use two data sets for phylogenetic analysis: the first consisting of novel sequences generated for this project and the second of additional, previously published sequences from the fruit- dove genus (Ptilinopus). Our analyses found support for the monophyly of a clade that contains a large fraction of the genera currently classified within Raphinae and also found several well-supported clades within this group of pigeons and doves. Character reconstruction methods based on the resulting phylogeny recover multiple transitions from a terrestrial to an arboreal foraging mode and evidence for multiple dispersal events from Asia to Africa throughout the history of the clade. Copyright 2019 by the authors. Published by the Illinois Natural History Survey under the terms of the creative commons attribution license http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. International • Peer-Reviewed • Open-Access Published continuously since 1876 Illinois Natural History Survey Bulletin 2 ILLINOIS NATURAL HISTORY SURVEY BULLETIN 42:2019001 Introduction Pigeons and doves (Aves: Columbidae) are a high- ly successful and diverse group of birds that are globally distributed and inhabit a variety of habi- tats (Goodwin 1983; Gibbs et al. 2001). However, despite publication of several studies on phyloge- netic relationships of Columbiformes, there are still many uncertainties about the diversification pat- terns within the order (Johnson and Clayton 2000a and 2000b; Johnson et al. 2001; Pereira et al. 2007; Gibb and Penny 2010; Johnson et al. 2010; Johnson and Weckstein 2011; Cibois et al. 2014; Sweet and Johnson 2015). One group that remains ambiguous is the fruit pigeons and doves (“fruit doves” here- after) and allied genera (Ptilinopus, Ducula, Gym- nophaps, Lopholaimus, Hemiphaga, Phapitreron, Goura, Caloenas, Otidiphaps, Trugon, Turtur, Oena, Chalcophaps, and Treron), which had previously been considered to be members of a subfamily, the Treroninae (del Hoyo et al. 1997). However, a more recent classification (del Hoyo et al. 2014) places all of these genera within an expanded group, the Raphinae, upon the discovery that the extinct dodo (Raphus cucullatus) is phylogenetically embedded within Treroninae (Shapiro et al. 2002; Pereira et al. 2007), and with Raphinae being the oldest available name for this group, as discussed by Cracraft in Dickinson and Remsen (2013). Pereira et al. (2007) identified the monophyly and composition of this group, but the result was somewhat unstable across analyses and included only limited taxon sampling. In addition to the more typical and large genera of fruit doves (Ptilinopus, Ducula, and Treron), several less diverse genera are associated with the fruit doves and allies. Del Hoyo et al. (1997) defined Treroninae as including the green pigeons (Treron), long-tailed pigeons (Gymnophaps, Cryptophaps, Lopholaimus, and Hemiphaga), fruit doves (Ducu- la, Alectroenas, Drepanoptila, and Ptilinopus), and brown pigeons (Phapitreron). The molecular phy- logeny of Pereira et al. (2007) also included wood doves (Turtur, Oena, and Chalcophaps), ground pi- geons (Otidiphaps, Trugon, Didunculus, Microgou- ra, Goura, and Caloenas), cloven-feathered doves (Drepanoptila), and blue pigeons (Alectroenas) in a clade with the Treroninae genera of del Hoyo et al. (1997). However, Gibbs et al. (2001) considered the wood doves (Chalcophaps, in particular) to be more closely related to bronzewings (Henicophaps) in the phabine clade (Phaps, Geophaps, Ocyphaps, Petrophassa, Geopelia, and Leucosarcia). Goodwin (1983) likewise considered Chalcophaps, along with Oena and Turtur, to be closely related to the phabines. He also included green pigeons, long- tailed pigeons, fruit doves, blue pigeons, and the cloven-feathered doves as a clade. Recently, del Hoyo et al. (2014) removed Treroninae from their classification and instead included all proposed Treroninae genera in an expanded subfamily Raph- inae. This classification corresponds to “Clade C” from Pereira et al. (2007) and is expanded to in- clude the phabine genera and allies. However, the inclusion of the phabines in this group was quite unstable. Shapiro et al. (2002) first recovered Dre- panoptila and Alectroenas nested within Ptilinopus, a relationship that has remained consistent in more recent studies (Cibois et al. 2014). This study had an extensive representation of the diverse Ptilinopus ge- nus but did not focus on other related genera. Given the recent instability in these classification schemes, it is important to sample genera and species more densely to identify stable phylogenetic patterns. The phylogenetic and taxonomic statuses of these lineages has important implications for the evolu- tion of Columbiformes, because this group could encompass a geographically and ecologically di- verse subset of taxa (Goodwin 1983; Gibbs et al. 2001; del Hoyo et al. 2014). The most species-rich ge- nus within this group, Ptilinopus, is found primarily in forest canopies of Southeast Asia and Oceania. Species in Ptilinopus vary in size but are all pri- marily frugivorous. Species from two other diverse genera, Ducula and Treron, are also primarily found in forest canopies and forage on fruit. However, Treron has a broad geographic range, with repre- sentatives in Asia and Africa. Additional arboreal and frugivorous groups with phylogenetic affinities likely include the long-tailed pigeons from Austral- asia (Gymnophaps, Lophalamius, and Hemiphaga), Phapitreron from the Philippines, Drepanoptila and Cryptophaps from Oceania, and Alectroenas from islands in the western Indian Ocean. Other allied genera are terrestrial and primarily granivorous. The large ground pigeons are terrestrial and found in rainforest habitats in Oceania. Shapiro et al. (2002) place the terrestrial Caloenas nicobarica (Nicobar pigeon) as the closest living relative to the extinct dodo (Raphus cucullatus). The small-bodied wood doves also forage on the ground and are distrib- uted in Africa and Australasia. Thus, understanding the transitions between terrestrial and arboreal for- aging requires more-detailed understanding of the phylogenetic relationships among these genera. Some studies have indicated that increased taxon sampling can help resolve phylogenetic relation- ships (Pollock et al. 2002; Hedtek et al. 2006; Prum et al. 2015), so here we include dense sampling to improve the ability to resolve the phylogenetic re- lationships within the fruit doves and their allies, focusing in particular on species from many of the genera mentioned. The taxa included in such a clade would provide insight into the taxonomic composition and phylogenetic structure to help NOWAK / PHYLOGENY OF FRUIT DOVES AND THEIR ALLIES 3 guide future subfamilial classifications. To address this question, we use multiple mitochondrial and nuclear genes for phylogenetic reconstruction. In addition to novel-sequence data, we also perform analyses combining newly collected sequence data with previously published data to provide a more comprehensive phylogeny of this group. Because these lineages of pigeons and doves are ecologi- cally diverse, we use the resulting phylogenies to provide insight into diversification patterns through ancestral state reconstruction of feeding mode and biogeographic areas. Methods Samples We sampled representatives from 14 genera (of 17 extant genera) and 45 species (of 155 extant species) of fruit doves and allies (Table 1). We also sampled 15 outgroup species from the three major clades of Columbiformes identified by Pereira et al. (2007), including multiple representatives from each clade. We rooted the tree on Clade A (Columba, Streptopelia, Patagioenas, Macropygia, Turacoena, Geotrygon, and Leptotila) identified by Pereira et al. (2007), because this group is consistently sepa- rated from members of Raphinae and/or Treroninae across all studies and classification schemes. These Clade A genera are part of the subfamily Columbi- nae, according to del Hoyo et al. (2014). DNA extraction, amplification, and sequencing We extracted DNA from feather and tissue sam- ples of wild or captive birds using a Qiagen Blood and Tissue Kit (Qiagen, Valencia, California, USA). Using polymerase chain reaction (PCR), we ampli- fied three mitochondrial loci: cytochrome oxidase subunit 1 (COI), cytochrome b (Cytb), and NADH dehydrogenase subunit 2 (ND2), and two nuclear loci: beta-fibrinogen introns 7 (FIB7) and 5 (FIB5). We used primers L6625 and H7005 to amplify COI (Hafner et al. 1994), primers L14841 and H4a (Koch- er et al. 1989) to amplify Cytb, primers L5215 and H6313 (Johnson and Sorenson 1998) to amplify ND2, primers FIBB17U and FIBB17L (Prychitko and Moore 1997) to amplify FIB7, and primers FIB5L and FIB6H (Marini and Hackett, 2002) to amplify FIB5. For sequencing we used the primers from the amplifi- cations; for larger genes we also used the following internal sequencing primers: L15517 and H15299 for Cytb (Harshman 1996), L5758s and H5766s for ND2 (Price et al. 2004), FIBDOVEF and FIBDOVER for FIB7 (Johnson and Clayton 2000a), and FIB-P4H and FIB-P3L for FIB5 (Cibois et al. 2014). We amplified selected loci with PCR according to previously used protocols for each locus (Johnson 2004; Pereira et al. 2007; Marini and Hackett 2002). We purified resulting PCR products using a Qiagen PCR Purification kit (Valencia, California, USA), and sequenced them using ABI Prism BigDye Ter- minators and Sanger DNA sequencing on an ABI 3730xl DNA Analyzer (University of Illinois Roy J. Carver Biotechnology Center, Champaign, Illi- nois, USA). We resolved resulting complementary chromatograms and trimmed primer sequences using Sequencher v. 5.0.1 (Gene Codes, Ann Arbor, Michigan, USA), and deposited all sequences in GenBank. We obtained additional sequences from GenBank to provide a more comprehensive data matrix with respect to taxon sampling, although fewer genes were available from published studies (see methods below). Phylogenetic analysis of complete five-gene data set For each of the five loci, we aligned all available sequences using MUSCLE (Edgar 2004) and visu- ally reviewed alignments in Seaview v. 4.2 (Gouy et al. 2010). To check for major discordances among gene trees, we constructed neighbor-joining and majority-rule maximum parsimony trees (100 ran- dom sampling replicates, Tree Bisection and Re- connection (TBR) branch swapping, 100 bootstrap replicates) for each gene separately using PAUP* v. 4.0b10 (Swofford 2003). With no major conflicts among gene trees, we proceeded to concatenate all loci using Seaview. Using the concatenated data set partitioned by locus, we used Bayesian and Maximum Likelihood (ML) mixed model analysis. We estimated appro- priate models for each locus using jModelTest2 (Akaike 1974; Darriba et al. 2012) based on the Akaike Information Criterion (AIC) values testing 88 different models. Model testing indicated that GTR+I+G models were best for mitochondrial loci (COI, Cytb, ND2) and GTR+G models were best for nuclear loci (FIB7 and FIB5). We ran ML analysis on our concatenated data set using Garli v. 2.0 (Zwickl 2006) with the aforemen- tioned gene-partition models and 500 bootstrap replicates, treating the mitochondrial genes as a single locus and the nuclear genes as two separate loci. We obtained a 50% majority-rule consensus tree from the bootstrap replicates using SumTrees (Sukumaran and Holder 2008). For Bayesian analy- sis, we used MrBayes v. 3.2 (Ronquist and Huelsen- beck 2003) on the CIPRES portal (Miller et al. 2010) with a mixed model analysis similar to our ML anal- ysis and default priors. We ran 4 runs with 4 chains for 20 million generations under MCMC sampling every 1,000 trees and viewed resulting trace files in Tracer v. 1.4 (Rambaut and Drummond 2007) to en- sure chain mixture and stationarity (ESS>200). We 4 ILLINOIS NATURAL HISTORY SURVEY BULLETIN 42:2019001 G EN U S SP EC IE S TI SS U E SO U RC E1 CO LL EC TI O N L O CA LI TY CY TB CO I N D 2 FI B 7 FI B 5 IN G RO U P C al o en as n ic o b ar ic a K U M N H B 15 80 ca p ti ve A F4 83 33 6 E F3 73 36 3 K T 02 34 02 K T 02 34 60 K T 02 98 57 C h al co p h ap s in d ic a FM N H 3 57 41 5 P h ili p p in es : M in d an ao A Y 44 36 72 K T 02 33 14 — — — C h al co p h ap s in d ic a A N W C 4 37 02 A u st ra lia : Q u ee n sl an d K T 02 33 65 K T 02 33 15 K T 02 34 03 K T 02 34 61 K T 02 98 57 C h al co p h ap s in d ic a T. P ra tt 2 00 3- 03 5 P ap u a N ew G u in ea : N o rm an b y Is . K T 02 33 64 K T 02 33 13 K T 02 34 04 K T 02 34 62 K T 02 98 58 C h al co p h ap s st ep h an i N M N H B 40 13 ca p ti ve A Y 44 36 73 E F3 73 36 5 K T 02 34 05 A Y 44 36 95 — C h al co p h ap s st ep h an i T. P ra tt 2 00 3- 02 6 P ap u a N ew G u in ea : N o rm an b y Is . K T 02 33 66 K T 02 33 16 — — K T 02 98 60 D u cu la b ak er i LS U B 45 40 9 Va n u at u : E sp ir it u S an to K T 02 33 69 K T 02 33 19 K T 02 34 08 K T 02 34 65 K T 02 98 64 D u cu la b ic o lo r LS U M N S B 19 21 7 ca p ti ve A F1 82 70 5 K T 02 33 21 K T 02 34 09 A F1 82 67 2 K T 02 98 65 D u cu la b ic o lo r A N W C 2 96 97 A u st ra lia : Q u ee n sl an d K T 02 33 70 K T 02 33 19 K T 02 34 10 K T 02 34 66 K T 02 98 66 D u cu la p ac ifi ca LS U B 45 43 1 Va n u at u : E sp ir it u S an to K T 02 33 71 K T 02 33 22 K T 02 34 11 K T 02 34 67 K T 02 98 67 D u cu la p ac ifi ca M K L3 S o lo m o n Is .: R en n el l K T 02 33 72 — — A Y 44 36 89 — D u cu la p in o n S m it h so n ia n –N S P Pa p u a N ew G u in ea : N o rm an b y Is . K T 02 33 73 K T 02 33 23 K T 02 34 12 K T 02 34 68 — D u cu la p is tr in ar ia U W B M 6 02 03 S o lo m o n Is .: K ia b a Is . A Y 44 36 69 K T 02 33 24 K T 02 34 13 A Y 44 36 91 K T 02 98 68 D u cu la ru b ic er a A M N H M K L 66 S o lo m o n Is .: Is ab el A Y 44 36 68 K T 02 33 25 — A Y 44 36 90 K T 02 98 69 D u cu la ru fi ga st er K U M N H 7 40 8 Pa p u a N ew G u in ea : W ab o K T 02 33 74 K T 02 33 26 K T 02 34 14 K T 02 34 69 K T 02 98 70 G o u ra cr is ta ta K U M N H B 15 88 ca p ti ve A F1 82 70 9 K T 02 33 27 K T 02 34 15 A F1 82 67 6 K T 02 98 71 G o u ra vi ct o ri a K U M N H 6 88 0 P ap u a N ew G u in ea : O ro P ro v. A F4 83 32 0 K T 02 33 28 K T 02 34 16 K T 02 34 70 K T 02 98 72 G ym n o p h ap s al b er ti si i LS U M N S B 28 85 6 ca p ti ve A Y 44 36 65 K T 02 33 29 K T 02 34 17 A Y 44 36 87 K T 02 98 73 H em ip h ag a n ov as ee la n d ia e fe at h er N ew Z ea la n d A Y 44 36 66 K T 02 33 30 K T 02 34 18 A Y 44 36 88 K T 02 98 74 Lo p h o la im u s an ta rc ti cu s A N W C 4 35 27 A u st ra lia E F3 73 28 2 K T 02 33 31 K T 02 34 19 E F3 73 48 5 K T 02 98 77 O en a ca p en si s LS U M N S B 34 20 7 S o u th A fr ic a A F1 82 70 7 E F3 73 38 3 E F3 73 34 5 A F1 82 67 4 — O en a ca p en si s FM N H 3 52 79 1 M ad ag as ca r K T 02 33 76 K T 02 33 33 K T 02 34 21 K T 02 34 72 K T 02 98 81 O ti d ip h ap s n o b ili s LS U M N S B 16 80 8 ca p ti ve A F4 83 35 2 K T 02 33 34 K T 02 34 22 E F3 73 48 7 K T 02 98 82 O ti d ip h ap s n o b ili s S E A 39 8 P ap u a N ew G u in ea : H er o w an a K T 02 33 77 K T 02 33 35 K T 02 34 23 K T 02 34 73 K T 02 98 83 P h ap it re ro n am et hy st in a FM N H A T P 92 -1 09 P h ili p p in es A F1 82 70 6 A F2 79 73 8 K T 02 34 24 A F1 82 67 3 K T 02 98 84 P h ap it re ro n am et hy st in a FM N H 3 92 23 2 P h ili p p in es : M in d an ao K T 02 33 78 E F3 73 38 7 — A F1 82 67 3 — P h ap it re ro n ci n er ei ce p s FM N H 3 57 41 0 P h ili p p in es K T 02 33 79 K T 02 33 38 K T 02 34 25 K T 02 34 74 K T 02 98 85 P h ap it re ro n le u co ti s FM N H 3 92 22 8 P h ili p p in es : M in d an ao A F2 79 71 2 A F2 79 73 9 — A F2 79 72 2 — P ti lin o p u s ci n ct u s LS U B 16 76 7 ca p ti ve K T 02 33 80 K T 02 33 38 K T 02 34 27 K T 02 34 76 K T 02 98 87 TA B LE 1 P ig eo n a n d d ov e sa m p le s u se d f o r a p hy lo g en et ic a n al ys is o f fr u it d ov es a n d t h ei r al lie s. NOWAK / PHYLOGENY OF FRUIT DOVES AND THEIR ALLIES 5 P ti lin o p u s co ro n u la tu s K U M N H 5 21 4 P ap u a N ew G u in ea : G u lf — K T 02 33 39 K T 02 34 28 K T 02 34 77 K T 02 98 88 P ti lin o p u s g re yi i LS U B 45 81 1 Va n u at u : E sp ir it u S an to K T 02 33 81 K T 02 33 40 K T 02 34 29 K T 02 34 78 K T 02 98 89 P ti lin o p u s io zo n u s LS U B 19 41 2 ca p ti ve — — — K T 02 34 79 K T 02 98 90 P ti lin o p u s le cl an ch er i FM N H 3 58 25 9 P h ili p p in es : S ib u ya n A F1 82 70 8 K T 02 33 41 K T 02 34 31 A F1 82 67 5 — P ti lin o p u s m ag n ifi cu s B W B 63 7 P ap u a N ew G u in ea : O ro P ro v. K T 02 33 83 K T 02 33 42 K T 02 34 32 K T 02 34 81 K T 02 98 91 P ti lin o p u s m el an o sp ila K U M N H B 15 81 ca p ti ve K T 02 33 84 — — — K T 02 98 92 P ti lin o p u s m el an o sp ila LS U M Z B 16 81 1 ca p ti ve K T 02 33 85 K T 02 33 43 K T 02 34 33 K T 02 34 82 K T 02 98 93 P ti lin o p u s o cc ip it al is FM N H 3 92 23 8 P h ili p p in es : M in d an ao A F2 79 71 3 A F2 79 74 0 K T 02 34 34 A F2 79 72 3 — P ti lin o p u s p er o u si i U W B M 4 28 42 To n ga K T 02 33 86 K T 02 33 44 K T 02 34 35 K T 02 34 83 K T 02 98 94 P ti lin o p u s p o rp hy re u s LS U B 14 20 9 ca p ti ve K T 02 33 87 K T 02 33 45 K T 02 34 36 K T 02 34 84 K T 02 98 96 P ti lin o p u s p o rp hy re u s FM N H 4 31 13 5 ca p ti ve K T 02 33 88 K T 02 33 46 K T 02 34 37 K T 02 34 85 K T 02 98 95 P ti lin o p u s p u lc h el lu s K U M N H 6 87 0 P ap u a N ew G u in ea : O ro P ro v. E F3 73 28 5 E F3 73 38 9 K T 02 34 38 K T 02 34 86 K T 02 98 97 P ti lin o p u s ra ro to n g en si s U W B M 4 25 45 C o o k Is .: A ti u A Y 44 36 63 K T 02 33 47 K T 02 34 39 A Y 44 36 85 K T 02 98 98 P ti lin o p u s re g in a A N W C 2 98 78 A u st ra lia : Q u ee n sl an d K T 02 33 89 K T 02 33 48 K T 02 34 40 K T 02 34 87 K T 02 98 99 P ti lin o p u s ri vo li K U M N H 7 41 2 P ap u a N ew G u in ea : H er o w an a K T 02 33 95 K T 02 33 54 K T 02 34 46 K T 02 34 93 K T 02 99 04 P ti lin o p u s ri vo li T. P ra tt 2 00 3- 10 0 P ap u a N ew G u in ea : F er g u ss o n Is . K T 02 33 90 K T 02 33 49 K T 02 34 41 K T 02 34 88 K T 02 99 00 P ti lin o p u s ri vo li K U M N H 4 74 5 P ap u a N ew G u in ea K T 02 33 91 K T 02 33 50 K T 02 34 42 K T 02 34 89 K T 02 99 01 P ti lin o p u s ri vo li K U M N H 4 77 1 P ap u a N ew G u in ea K T 02 33 92 K T 02 33 51 K T 02 34 43 K T 02 34 90 K T 02 99 02 P ti lin o p u s ri vo li K U M N H 4 78 8 P ap u a N ew G u in ea K T 02 33 93 K T 02 33 52 K T 02 34 44 K T 02 34 91 — P ti lin o p u s ri vo li K U M N H 4 80 5 P ap u a N ew G u in ea K T 02 33 94 K T 02 33 53 K T 02 34 45 K T 02 34 92 K T 02 99 03 P ti lin o p u s su p er b u s B W B 63 8 P ap u a N ew G u in ea : O ro P ro v. — K T 02 33 55 K T 02 34 47 K T 02 34 94 K T 02 99 05 Tr er o n ca lv a LS U B 45 25 6 G h an a K T 02 33 96 E F3 73 39 2 K T 02 34 48 K T 02 34 95 K T 02 99 07 Tr er o n ca lv a A M N H A LP 8 0 C en tr al A fr ic an R ep u b lic A Y 44 36 74 K T 02 33 56 K T 02 34 49 A Y 44 36 96 — Tr er o n si eb o ld ii LS U B 16 97 8 Ja p an : O sa ka P re fe ct u re K T 02 33 97 K T 02 33 57 K T 02 34 50 — K T 02 99 08 Tr er o n ve rn an s LS U M Z B 20 69 6 ca p ti ve A F4 83 32 1 K T 02 33 58 K T 02 34 52 A F1 82 67 7 K T 02 99 10 Tr er o n w aa lia FM N H 3 96 40 6 G h an a A F4 83 35 0 K T 02 33 59 K T 02 34 53 — K T 02 99 11 Tr u g o n te rr es tr is K U M N H B 51 00 Pa p u a N ew G u in ea : G u lf — — K T 02 34 51 K T 02 34 96 K T 02 99 09 Tu rt u r ab ys si n ic u s LS U M Z B 45 03 4 G h an a: N o rt h er n R eg io n K T 02 33 98 K T 02 33 60 K T 02 34 54 K T 02 34 97 K T 02 99 12 Tu rt u r ab ys si n ic u s fe at h er C am er o o n K T 02 33 99 K T 02 33 61 K T 02 34 55 — — Tu rt u r af er A M N H R W D 23 76 1 C en tr al A fr ic an R ep u b lic K T 02 34 00 K T 02 33 62 K T 02 34 56 — — Tu rt u r b re h m er i A M N H P R S 2 04 8 C en tr al A fr ic an R ep u b lic A Y 15 10 05 A Y 15 10 08 K T 02 34 57 A Y 15 10 06 — Tu rt u r ch al co sp ilo s LS U M Z B 34 26 3 S o u th A fr ic a A Y 44 36 71 E F3 73 39 5 E F3 73 35 7 A Y 44 36 93 — Tu rt u r ty m p an is tr ia FM N H 3 55 25 2 U ga n d a K T 02 34 01 — K T 02 34 59 K T 02 34 98 — (c o n ti n u ed ) 6 ILLINOIS NATURAL HISTORY SURVEY BULLETIN 42:2019001 G EN U S SP EC IE S TI SS U E SO U RC E1 CO LL EC TI O N L O CA LI TY CY TB CO I N D 2 FI B 7 FI B 5 O U TG RO U PS C la ra vi s m o n d et o u ra LS U B 16 22 1 C o st a R ic a K J6 39 09 3 K J6 30 87 8 K J6 45 73 6 K J6 68 68 6 K T 02 98 61 C o lu m b a ir id it o rq u es FM N H 4 86 84 2 M al aw i K T 02 33 67 K T 02 33 17 K T 02 34 06 K T 02 34 63 — C o lu m b a ru p es tr is U W B M 5 97 55 R u ss ia A F3 53 41 0 A F3 53 48 2 A F3 53 43 4 A F3 53 46 1 K T 02 98 63 C o lu m b in a p as se ri n a K U M N H B 17 55 U S A : M is so u ri K J6 39 10 2 K J6 30 88 7 K J6 45 74 5 K J6 68 69 5 — G eo tr yg o n co st ar ic en si s N M N H 0 15 44 Pa n am a A Y 44 36 60 H Q 99 35 29 H Q 99 35 49 A Y 44 36 82 — Le p to ti la ja m ai ce n si s K M N H 2 13 5 M ex ic o A F2 79 70 6 A F2 79 72 6 H Q 99 35 43 A F2 79 71 6 K T 02 98 75 Le u co sa rc ia m el an o le u ca A N W C 4 97 17 A u st ra lia — E F3 73 37 9 E F3 73 34 1 K T 02 34 71 K T 02 98 76 M ac ro p yg ia m ac ki n la yi A M N H M K L- 82 S o lo m o n s Is . A F3 53 41 5 A F3 53 49 0 A F3 53 44 4 A F3 53 46 6 K T 02 98 78 M ac ro p yg ia p h as ia n el la 29 21 19 A u st ra lia K T 02 33 75 K T 02 33 32 K T 02 34 20 A F1 82 66 0 K T 02 98 79 M et ri o p el ia m el an o p te ra K G M 44 3 A rg en ti n a K J6 39 09 2 K J6 30 87 7 K J6 45 73 5 K J6 68 69 2 K T 02 98 80 Pa ta g io en as p ic az u ro LS U 1 61 59 ca p ti ve K T 02 33 68 K T 02 33 18 K T 02 34 07 K T 02 34 64 K T 02 98 62 P h ap s el eg an s A N W C 4 28 7 A u st ra lia — K T 02 33 37 K T 02 34 26 K T 02 34 75 K T 02 98 86 S tr ep to p el ia o ri en ta lis U W B M 4 72 82 R u ss ia A F3 53 40 5 A F3 53 47 6 A F3 53 42 6 A F3 53 45 6 K T 02 99 06 Tu ra co en a m an ad en si s 16 95 9 ca p ti ve E F3 73 28 7 K T 02 33 63 K T 02 34 58 — K T 02 99 13 U ro p el ia ca m p es tr is LS U C C W 92 5 B o liv ia K J6 39 09 8 K J6 30 88 3 K J6 45 74 1 K J6 68 69 1 K T 02 99 14 TA B LE 1 P ig eo n a n d d ov e sa m p le s u se d f o r a p hy lo g en et ic a n al ys is o f fr u it d ov es a n d t h ei r al lie s (c o n ti n u ed ). N o te : A cc es si o n n u m b er s fo r se q u en ce s g en er at ed f ro m t h is s tu d y b eg in w it h K T. G en B an k ac ce ss io n n u m b er s in d ic at e su cc es sf u l s eq u en ce s. 1. M u se u m o r co lle ct io n v o u ch er n u m b er . NOWAK / PHYLOGENY OF FRUIT DOVES AND THEIR ALLIES 7 also assessed topological convergence between runs using AWTY (Wilgenbusch et al. 2004). Based on the trace files, ESS values, and AWTY results, we discarded the first 2,000 trees (10%) as a burn-in. We edited the resulting consensus trees in Figtree v. 1.4 (Rambaut 2012). Phylogenetic analysis with additional taxon sampling In addition to our own data, we obtained Gen- Bank sequences for loci ND2 and FIB5 (GenBank accession numbers KF446677- through KF446871) from some Ptilinopus, Alectroenas, Drepanoptila, Ducula, Treron, and Caloenas from a previously published study (Cibois et al. 2014) and combined them with our data to form a more taxonomically comprehensive data set (referred to throughout as the “combined data set”). Several sequences deposited in GenBank by Cibois et al. (2014) were generated from the same museum tissue samples that we used to generate our own data set. Thus, we did not include these GenBank sequences in our combined data set. Sequences for the other three genes for these additional taxa were not available, so we coded them as missing data. For this com- bined data set we carried out phylogenetic inference using the same methods as with our five-gene data set, aligning each locus with MUSCLE and checking each alignment by eye. We used both Bayesian and ML analyses on the combined concatenated data set. We once again tested for appropriate models at each locus using jModelTest2 and found AIC results identical to our smaller data set. We implemented ML and Bayesian analyses as with our complete five-gene data set, using Garli v. 2.0 and MrBayes v. 3.2, respectively. Ancestral state reconstruction Since we are interested in both biogeographic and ecological (foraging mode) patterns of evolution in this group of doves, we used both ancestral state– reconstruction and ancestral range–reconstruction methods. To reconstruct the ancestral state of forag- ing-mode transitions within this group, we inferred an ultrametric tree and used several ancestral state–reconstruction methods. We were interested in testing how many transitions in foraging mode occurred in this group. To infer an ultrametric tree, we used BEAST v. 1.8.1 (Drummond et al. 2012) on the CIPRES portal. We partitioned the data by locus, using the same models as those used in the Bayes- ian and ML analyses, a Yule tree prior, and strict branch-length priors with uniform distributions for each gene partition. We ran a single MCMC of 20 million generations, sampling every 1,000 trees and discarding the first 2,000 trees as burn-in. We confirmed that the run reached stationarity in Tracer by ensuring that the Effective Sample Size (ESS) values were >200, and we summarized the posterior distribution of post-burn-in trees with a maximum clade credibility tree generated in Tree- Annotator v. 1.8.1. We then coded each tip as either an arboreal or terrestrial forager according to Gibbs et al. (2001) and Goodwin (1983) and mapped the character state reconstruction onto the ultrametric tree using both parsimony and likelihood recon- struction methods in Mesquite v. 2.75 (Maddison and Maddison 2015). We used the MK1 model in the likelihood reconstruction. We also used a Bayesian reconstruction method—Bayesian binary MCMC (BBM)—as implemented in Reconstruct Ances- tral State in Phylogenies (RASP) v. 3.0 (Yu et al. 2014). Although this method is primarily intended to reconstruct biogeographic scenarios, it is also appropriate for use in character reconstruction of binary characters. Allowing for one maximum state at each node (BBM allows for the possibility of mul- tiple state probabilities at each ancestral node), we ran the MCMC analysis for 10 chains of 5 million generations, sampling every 1,000 generations and discarding the first 500 samples as a burn-in. For each of the three analyses (parsimony, likelihood, and BBM), we used data sets with and without the outgroup taxa included to test for biases in results due to outgroup character states. For ancestral range reconstruction, we used BBM implemented in RASP. Because RASP can use mul- tiple trees to account for phylogenetic uncertainty, we input the post-burn-in posterior distribution of BEAST trees and removed outgroup and dupli- cate (conspecific) taxa. We then coded each taxon as having an Asian or African range distribution. We randomly sampled 1,000 BEAST trees using the RASP interface and ran BBM allowing for two maximum states at each node. We set the MCMC analysis parameters to run 10 chains of 5,000,000 cycles, sampling every 100 samples and discarding the first 100 samples as a burn-in. Results Phylogenetic analysis The final concatenated data set collected by us was 4,277 aligned base pairs from a total of 77 different individual samples (Table 1). We obtained sequence data for each of the 5 loci for the majority of the ingroup and outgroup samples, with an 88% complete matrix (obtained sequence data/possible sequence data). Both the ML and Bayesian analy- ses generated similar trees (Figure 1), and a large percentage of ingroup nodes from both the ML tree (~75%) and the Bayesian tree (~79%) received high support values (≥90% maximum likelihood boot- strap replicates [ML]/≥0.95 posterior probability 8 ILLINOIS NATURAL HISTORY SURVEY BULLETIN 42:2019001 [PP]). The Bayesian analysis provided strong sup- port (1.0 PP) for monophyly of the group containing all of the previously recognized genera allied with the fruit doves, exclusive of the phabines, and the ML analysis provided modest support (68 ML) for this relationship. We suggest this is the clade that should designate the Raphinae, because it is rela- tively well supported, containing genera previously placed within either Treroninae or Raphinae. In ad- dition, further expansion of this clade lacks support. In addition to defining a major clade of fruit doves and allied genera, there is high support from both analyses for several subclades within the ingroup. The genera Ptilinopus and Ducula form reciprocal- ly monophyletic groups (100 ML/1.0 PP for both Ptilinopus and Ducula). However, the relation- ship between the two clades is unclear, with low support of a sister relationship in both analyses (52 ML/0.85 PP). Lopholaimus, Gymnophaps, and Hemiphaga also form a well-supported clade (100 ML/1.0 PP), and together with Ptilinopus and Ducula form a clade (95 ML/1.0 PP). Other well-supported monophyletic genera (not including genera with a single representative species) are Phapitreron, Goura, Otidiphaps, Turtur, Chalcophaps, and Treron. Among these genera, Goura has support as being sister to the monotypic genus Caloenas, whereas Trugon and Otidiphaps have moderate support as being sister taxa (72 ML/1.0 PP). Turtur is supported as sister to the monotypic genus Oena (100 ML/1.0 PP), and together with Chalcophaps form a clade (100 ML/1.0 PP). Species of the genus Treron have support as being sister to the rest of the ingroup in the Bayesian analysis (1.0 PP) but not in the ML (<50 ML) analysis. Given our limited within-species sampling, we recovered all species as monophylet- ic with the exception of Treron calva. Treron waalia is nested within T. calva (93 ML/1.0 PP). The combined data set, including our data and data from Cibois et al. (2014), resulted in a total of 204 samples. As with the previous complete five-gene data set, a high fraction of nodes were strongly sup- ported (≥90% ML/≥0.95 PP) by both the ML (~55%) and Bayesian (~85%) analyses (Supplementary Figure S1). The combined data analysis is generally consistent with the results of analysis on our five- gene data set. With this expanded taxon sampling, the species Ptilinopus purpuratus, P. porphyraceus, P. mericierii, and P. viridis (in addition to Treron calva) are not monophyletic. Ptilinopus and the embedded genera Alectroenas and Drepanoptila have strong support as a monophyletic group (100 ML/1.0 PP). We recover Ducula as being monophy- letic (100 ML/1.0 PP), but its relationship as sister to the Ptilinopus clade is not well supported. Ancestral character state reconstruction and biogeographic analysis The parsimony, likelihood, and Bayesian ancestral character reconstruction methods of foraging mode all produced very similar results (Figure 2). In the analysis that included outgroups, all three methods recovered a terrestrial foraging ancestral state for the fruit-doves-and-allies clade, with two indepen- dent transitions to an arboreal foraging state. We estimated that these transitions occurred along the branch leading to the Treron clade and again along the branch leading to the Ptilinopus + Ducula + Lopholaimus + Gymnophaps + Hemiphaga clade. Using all three ancestral character state–reconstruc- tion methods, our character state–reconstruction analysis without outgroups also indicated multiple transitions in foraging mode, although we were un- able to confidently determine directionality of these transitions (Supplementary Figure S2). The Bayesian ancestral range reconstruction method (BBM) recovered an Asian ancestral range for this group (1.0 probability). The analysis also recovered two independent dispersal events from Asia to Africa (Figure 2). One dispersal event was recovered at the ancestral node of Chalcophaps (an Asian genus) and Turtur and Oena (both African genera). The second dispersal event was recovered at the ancestral node of Treron vernans (an Asian species) and Treron calva, and Treron waalia (both African species). Discussion Identification of a monophyletic clade among the fruit doves and allies Phylogenetic relationships of fruit doves and their allies based on five molecular loci are generally well supported. They largely agree with previous, less exhaustive phylogenetic analyses (Johnson et al. 2001; Pereira et al. 2007; Gibb and Penny 2010; Cibois et al. 2014), although important distinctions exist. Perhaps most notable, we recovered a clade of fruit doves and allies as monophyletic with high support in most of our analyses, which includes most, but not all, of the genera currently classified in the Raphinae by del Hoyo et al. (2014). This major clade was poorly resolved in previous studies, in particular because of weakly supported deeper-lev- el relationships within Columbiformes. Pereira et al. (2007) placed the Australian phabine clade (rep- resented in our data set by Phaps and Leucosarcia) as sister to a poorly supported fruit-doves-and-al- lies clade. Gibb and Penny (2010) do not recover a monophyletic fruit-doves-and-allies clade, placing small New World ground doves (subfamily Clar- avinae) within the clade. However, our analysis places the small New World ground doves clearly NOWAK / PHYLOGENY OF FRUIT DOVES AND THEIR ALLIES 9 1 1 0.03 substitutions/site Ptilinopus rivoli 2003-100 Ptilinopus leclancheri 358259 Ducula pinon 2003-036 Caloenas nicobarica B1580 Turtur afer RWD23761 Ptilinopus coronulatus 5214 Columbina passerina Uropelia campestris Chalcophaps stephani 2003-026 Chalcophaps indica 357415 Leucosarcia melanoleuca Phapitreron amethystina 392232 Ptilinopus pulchellus 6870 Patagioenas picazuro Ptilinopus rivoli 7412 Otidiphaps nobilis SEA398 Ptilinopus occipitalis 392238 Turtur tympanistria 355252 Ptilinopus rivoli 4788 Goura cristata B1588 Columba rupestris Treron calva B45256 Leptotila jamaicensis Ptilinopus rivoli 4771 Turacoena manadensis Geotrygon costaricensis Ptilinopus magnificus BWB637 Macropygia mackinlayi Ptilinopus rarotongensis 42545 Ducula pistrinaria 60203 Chalcophaps indica 2003-035 Ptilinopus perousii 42842 Ptilinopus greyii B45811 Turtur abyssinicus B45034 Otidiphaps nobilis B16808 Ducula bakeri B45409 Phapitreron cinereiceps 357410 Oena capensis 352791 Ducula rufigaster 7408 Treron calva ALP80 Ducula pacifica B45431 Ptilinopus porphyreus B14209 Ducula rubricera MKL66 Metriopelia melanoptera Treron vernans B20696 Oena capensis B34207 Chalcophaps indica 43702 Phaps elegans Ducula bicolor 29697 Chalcophaps stephani B4013 Ptilinopus iozonus B19412 Turtur brehmeri PRS2048 Ptilinopus regina 29878 Ptilinopus superbus BWB638 Goura victoria 6880 Claravis mondetoura Lopholaimus antarcticus 43527 Turtur chalcospilos B34263 Treron sieboldii B16978 Treron waalia 396406 Ptilinopus rivoli 4805 Ptilinopus cinctus B16767 Turtur abyssinicus Ptilinopus porphyreus 431135 Phapitreron amethystina ATP92-109 Ducula pacifica MKL3 Macropygia phasianella Ptilinopus rivoli 4745 Phapitreron leucotis 392228 Trugon terrestris B5100 Streptopelia orientalis Ducula bicolor B19217 Gymnophaps albertisii B28856 Hemiphaga novaeseelandiae Ptilinopus melanospila B16811 Columba iriditorques 84/0.99 99/1.0 52/0.85 54/0.71 -/1.0 73/0.86 53/1.0 72/1.0 79/0.92 73/0.95 98/1.0 -/0.69 100/1.0 -/0.77 -/0.86 93/1.0 -/0.71 61/0.66 100/1.0 -/0.66 72/1.0 71/0.85 -/0.92 -/0.90 ML/PP 97/1.0 99/1.0 95/1.0100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 92/1.0 100/1.0 97/1.0 100/1.0 95/1.0 100/1.0 100/1.0 100/1.0 63/1.0 100/1.0 -/0.89 99/1.0 100/1.0 100/1.0 99/1.0 100/1.0 100/1.0 99/1.0 100/1.0 100/1.0 100/1.0 68/1.0 100/1.0 61/1.0 100/1.0 94/1.0 100/1.0 100/1.0 100/1.0 -/0.51 100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 100/1.0 R ap hi na e Claravinae phabines Columbinae FIGURE 1 Bayesian 50% majority-rule consensus tree of fruit doves and their allies. Numbers at each node indicate the bootstrap and posterior probability values. Dashes indicate bootstrap values <50. Letters and numbers after each taxon name refer to the specific tissue voucher numbers in Table 1. Relevant higher taxonomic groups are indicated to the right of the tip names. The scale bar indicates the rate of nucleotide substitutions per site. 10 ILLINOIS NATURAL HISTORY SURVEY BULLETIN 42:2019001 Ptilinopus rivoli 2003-100 Ptilinopus leclancheri 358259 Ducula pinon 2003-036 Caloenas nicobarica B1580 Turtur afer RWD23761 Ptilinopus coronulatus 5214 Columbina passerina Uropelia campestris Chalcophaps stephani 2003-026 Chalcophaps indica 357415 Leucosarcia melanoleuca Phapitreron amethystina 392232 Ptilinopus pulchellus 6870 Patagioenas picazuro Ptilinopus rivoli 7412 Otidiphaps nobilis SEA398 Ptilinopus occipitalis 392238 Turtur tympanistria 355252 Ptilinopus rivoli 4788 Goura cristata B1588 Columba rupestris Treron calva B45256 Leptotila jamaicensis Ptilinopus rivoli 4771 Turacoena manadensis Geotrygon costaricensis Ptilinopus magnificus BWB637 Macropygia mackinlayi Ptilinopus rarotongensis 42545 Ducula pistrinaria 60203 Chalcophaps indica 2003-035 Ptilinopus perousii 42842 Ptilinopus greyii B45811 Turtur abyssinicus B45034 Otidiphaps nobilis B16808 Ducula bakeri B45409 Phapitreron cinereiceps 357410 Oena capensis 352791 Ducula rufigaster 7408 Treron calva ALP80 Ducula pacifica B45431 Ptilinopus porphyreus B14209 Ducula rubricera MKL66 Metriopelia melanoptera Treron vernans B20696 Oena capensis B34207 Chalcophaps indica 43702 Phaps elegans Ducula bicolor 29697 Chalcophaps stephani B4013 Ptilinopus iozonus B19412 Turtur brehmeri PRS2048 Ptilinopus regina 29878 Ptilinopus superbus BWB638 Goura victoria 6880 Claravis mondetoura Lopholaimus antarcticus 43527 Turtur chalcospilos B34263 Treron sieboldii B16978 Treron waalia 396406 Ptilinopus rivoli 4805 Ptilinopus cinctus B16767 Turtur abyssinicus Ptilinopus porphyreus 431135 Phapitreron amethystina ATP92-109 Ducula pacifica MKL3 Macropygia phasianella Ptilinopus rivoli 4745 Phapitreron leucotis 392228 Trugon terrestris B5100 Streptopelia orientalis Ducula bicolor B19217 Gymnophaps albertisii B28856 Hemiphaga novaeseelandiae Ptilinopus melanospila B16811 Columba iriditorques Africa 0.29 0.03 0.97 0.97 0.98 0.98 0.92 0.98 0.99 0.97 0.95 0.03 0.01 0.60 0.010.53 0.54 0.02 0.97 0.86 0.78 0.99 0.98 Dispersal Dispersal R ap hi na e Africa NOWAK / PHYLOGENY OF FRUIT DOVES AND THEIR ALLIES 11 outside the group. Our Bayesian analysis recovers a well-supported fruit-doves-and-allies clade (1.0 PP), although bootstrap support by ML analysis is more moderate (68 ML). Both Bayesian and ML analyses of our combined data set also supports the clade (76 ML/1.0 PP). Our overall results are most similar to Pereira et al. (2007), but we had higher support for a fruit-doves-and-allies clade. Although we analyzed fewer loci than Pereira et al. (2007) we had much denser taxonomic sampling, which likely contributed to higher resolution (Pollock et al. 2002; Hedtek et al. 2006). Based on these results and previously published studies, we suggest the Raphinae should be modified from del Hoyo et al. (2014) to include the genera Trugon, Otidiphaps, Microgoura (extinct), Goura, Caloenas, Raphus (extinct), Pezophaps (extinct), Chalcophaps, Turtur, Oena, Phapitreron, Treron, Ducula, Ptilinopus, Alec- troenas, Drepanoptila, Hemiphaga, Cryptophaps, Gymnophaps, and Lopholaimus. Genera in the phabine clade should not be included in Raphinae. Phylogenetic relationships within and among genera All genera except Ptilinopus are supported as monophyletic. Inclusion of the data from Cibois et al. (2014) reveals that the genera Drepanoptila and Alectroenas are embedded within Ptilinopus. Drepanoptila and Alectroenas were not sampled in the five-gene data set, so this result could not be tested using all five genes. However, this result is also consistent with the phylogeny reported by Gibb and Penny (2010). Based on these previous studies, del Hoyo et al. (2014) split Ptilinopus into multiple genera, but the other option is to subsume the smaller genera Drepanoptila and Alectroenas into Ptilinopus. With the combined data set, four additional species were recovered as paraphyletic: Ptilinopus purpuratus, P. porphyraceus, P. mericie- rii, and P. viridis. Analysis from Cibois et al. (2014) also recovered P. purpuratus and P. porphyraceus as paraphyletic. Ptilinopus viridis is rendered paraphyletic by the insertion of a closely related sister taxon, P. eugeniae, although this relationship is not well supported (<50 ML/0.54 PP). Two speci- mens of P. mericierii are recovered as closer to the sister species P. dupetithouarsii than to a third P. mericierii specimen, but this is also not well sup- ported (<50 ML/0.68 PP). Cibois et al. (2014) recover P. mericierii and P. dupetithouarsii as reciprocally monophyletic but with low Bayesian support for the monophyly of P. mericierii. Similar to prior studies, we recover Ptilinopus, Ducula, and the long-tailed pigeons (Gymnophaps, Lopholaimus, and Hemiphaga) as a clade, although the relationships among these genera are not com- pletely clear. Other studies have placed long-tailed pigeons as sister to Ptilinopus (Pereira et al. 2007; Gibb and Penny 2010), but these results were not well supported. In contrast, with relatively weak support we recovered Ducula as sister to Ptilino- pus. Similar to our results, studies by Shapiro et al. (2002) and Cibois et al. (2014) place Ducula sister to Ptilinopus, although these studies did not have ex- tensive sampling, with only three species of Ducula in each study. Future work may require additional nuclear data to elucidate the deeper relationships within this clade. We also recover Turtur, Oena, and Chalcophaps as a well-supported clade. A study by Khan and Arif (2013) found similar results using the mitochondrial locus COI. Our results place Turtur sister to Oena, with this clade sister to Chalcophaps. This agrees with studies by Pereira et al. (2007) and Shapiro et al. (2002). Gibb and Penny (2010) also recovered Oena and Chalcophaps as sister taxa, al- though they did not include Turtur in their analysis. Our phylogeny also places Goura sister to Caloenas, and Otidiphaps sister to Trugon. However, only the Bayesian analysis recovers all four of these genera together as a clade. Gibb and Penny’s (2010) phylog- eny also places Goura sister to Caloenas; however, Trugon is not included in their analysis. They placed Goura and Caloenas sister to Otidiphaps. Finally, we recover Treron—the green pigeons—as sister FIGURE 2 An ultrametric phylogeny for fruit doves and their allies. Taxon names are the same as in Figure 1, with letters and numbers following each name referencing the specific tissue vouchers in Table 1. Branch colors indicate a parsimony reconstruction analysis in Mesquite. Brown branches indicate a terrestrial foraging mode, and green branches indicate an arboreal foraging mode. Values above nodes are the proportional likelihood values from the likelihood reconstruction analysis in Mesquite. Values are from 0 to 1 and indicate the likelihood a particular ancestral node was a terrestrial forager. Nodes without values indicate support for a foraging mode >0.99 and agree with the parsimony results (e.g., a node without associated values that bifurcates into two brown branches has >0.99 support for a terrestrial foraging mode, and vice versa on nodes bifurcating into two black branches). Values listed below the nodes are posterior probability values from a Bayesian MCMC (BBM) reconstruction analysis in RASP. Scale and interpretation are the same as the likelihood results. Pie charts indicate the ancestral range reconstruction from the BBM model. Red indicates the probability of an African ancestral range, blue indicates the probability of an Asian ancestral range, and yellow indicates the probability of an ancestral range in both Africa and Asia. Nodes with probability values >0.95 for an Asian ancestral range are not shown. Arrows indicate probable dispersal events. 12 ILLINOIS NATURAL HISTORY SURVEY BULLETIN 42:2019001 to the rest of the fruit-doves-and-allies clade in our Bayesian analysis (1.0 PP). However, this relation- ship is unresolved in our ML analysis. Analyses of our combined dataset gives similar results, with Treron recovered as sister to the remainder of the fruit doves and allies in the Bayesian analysis but as unresolved in the ML analysis. Therefore, we are unable to as confidently resolve the placement of Treron with either data set. Although the Bayesian results may be correct in placing the genus as sister to the other ingroup taxa, additional data are need- ed to confirm this relationship. Additionally, Treron calva is the only species recovered as paraphyletic in our analysis, with T. waalia nested within the T. calva clade. This relationship perhaps indicates recent speciation within the genus Treron due to biogeographic dispersal from Asia to Africa. Multiple foraging transitions All three of ancestral character reconstruction meth- ods (parsimony, likelihood, and BBM) recover multiple, independent transitions in foraging mode within the fruit doves and allies. We obtain this result in separate sets of analyses with and with- out the outgroup taxa included. Although we are no longer able to confidently identify the directionality of the foraging transitions (i.e., terrestrial to arboreal, or vice versa) when we remove the outgroup, the recon- structions still retain multiple independent transition combinations (Supplementary Figure S2). When the outgroup is included, all three of the character reconstruction methods recover terres- trial foraging as the ancestral state for the fruit- doves-and-allies clade (Figure 2). This result is well supported in both the likelihood and Bayesian MCMC analyses. Many species of doves eat seeds and/or fallen fruit from the ground, and these re- sults indicate that at least the common ancestors of the taxa in this analysis were perhaps granivorous terrestrial foragers. These results also suggest that the mostly frugivorous, arboreal foraging habit is a more derived state, thus suggesting that the ancestors of fruit doves and allies may have been primarily terrestrial. Many of the contemporary granivorous, terrestrial foraging doves live in areas dominated by scrubby vegetation and/or grasses (Goodwin 1983; Gibbs et al. 2001). The transitions from terrestrial to arboreal foraging would there- fore also indicate a transition in habitat, from scrub- by or grassland areas to more heavily forested areas, where fruit would be available in the canopy. Several terrestrial foragers do not live in scrubby or open habitat, however, and in fact prefer heav- ily forested rainforest habitats. For example, the pheasant pigeon (Otidiphaps nobilis) is a terrestrial pigeon from rainforests of New Guinea and nearby islands (Gibbs et al. 2001). Crowned pigeons (in the genus Goura) live in similar habitats. These larger ground-foraging pigeons often eat fallen fruits as well as seeds (Pratt et al. 2015). Additionally, some arbo- real foragers prefer open habitat. For example, the orange-fronted fruit dove (Ptilinopus aurantiifrons) forages on fruit in the canopy but primarily lives in more open areas of New Guinea (Pratt et al. 2015). Multiple dispersal events into Africa Our biogeographic analysis recovered two disper- sal events from Asia to Africa within the fruit doves and allies clade (Figure 2). Dispersal into Africa likely occurred within the Chalcophaps, Turtur, and Oena clade and the Treron vernans, Treron clava, and Treron waalia clade. Turtur and Oena are Af- rican genera, whereas species of Chalcophaps are distributed in Asia and Australasia. Chalcophaps is the sister group to Turtur and Oena, and the three genera share a relatively deep ancestral node in the phylogeny. This perhaps indicates a more ancient dispersal into Africa. However, dispersal of Treron between Asia and Africa likely occurred much later. Most species of Treron have ranges in eastern Asia, whereas T. calva and T. waalia are native to sub-Sa- haran Africa. The African species of Treron from this study are separated from the Asia species by a relatively short branch, suggesting that this was a more recent dispersal event from Asia to Africa. Furthermore, the short branches among T. calva and T. waalia specimens indicate recent speciation within the African Treron. This is perhaps evidence of subsequent radiation once Treron dispersed into Africa. It would be interesting to include the other African Treron species (T. delalandii, T. griveaudi, T. sanctihomae, T. pembaensis, and T. australis) in a similar phylogenetic analysis to estimate branch lengths and genetic distances and to assess mono- phyly among those taxa. Conclusion From an extensive sampling of fruit doves and al- lied genera, we estimated a phylogeny of these taxa from both nuclear and mitochondrial DNA sequenc- es. We found support for seven major clades, as well as identified a clade that we feel could be more con- fidently defined as the subfamily Raphinae (having priority over Treroninae) within Columbidae. The status of proposed members of this subfamily has been unclear in previously published, family-wide phylogenies of pigeons and doves (e.g., Pereira et al. 2007). These previously published studies pro- vided great insight into many of the phylogenetic relationships within Columbidae. However, they NOWAK / PHYLOGENY OF FRUIT DOVES AND THEIR ALLIES 13 did not have sufficiently broad taxonomic sampling of the fruit-doves-and-allies clade to represent the taxonomic diversity of this group. Here we used a data set with many representatives from through- out the clade to clarify its status within Columbidae. Having established many of the phylogenetic pat- terns among fruit doves and their allies, we were able to address some questions related to the evo- lutionary history of the group. Since members of the fruit-doves-and-allies clade include both terres- trial and arboreal foragers, we evaluated transitions between these two foraging modes and found evi- dence for multiple transitions. In our analysis with outgroup taxa, we recovered terrestrial foraging as the ancestral state with two transitions to arboreal foraging. Additionally, we evaluated broad biogeo- graphic patterns in the group. Our ancestral range reconstruction indicated two separate dispersal events from Asia into Africa. Acknowledgments We thank Therese Catanach, Julie Allen, and Patrick Gero in helping to provide feedback and assistance with the phylogenetic analyses. We also thank Mi- chael Andersen for helpful comments that improved the manuscript. We thank the following individuals and institutions for providing tissue samples: Field Museum of Natural History, Kansas University Museum of Natural History Survey, University of Washington Burke Museum, American Museum of Natural History, Louisiana State University Muse- um of Natural Science, Australian National Wildlife Collection of the Commonwealth Scientific and Industrial Research Organisation, Sarah Bush, Brett Benz, Martyn Kennedy, and Thane Pratt. This research was supported by National Science Foundation grants DEB-0118794, DEB-0612938, DEB-1050706, DEB-1239788, and DEB-1342604 to Kevin P. Johnson. 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