Molecular Phylogenetics and Evolution 56 (2010) 393–408Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Neotropical mutualism between Acacia and Pseudomyrmex: Phylogeny and divergence times Sandra Gómez-Acevedo a,*, Lourdes Rico-Arce b, Alfonso Delgado-Salinas c, Susana Magallón c, Luis E. Eguiarte a a b c Laboratorio de Evolución Molecular y Experimental, Departamento de Ecología Evolutiva, Instituto de Ecología, UNAM. Apartado Postal 70-275, 04510 México, D.F., Mexico HLAA, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK Departamento de Botánica, Instituto de Biología, UNAM. Apartado Postal 70-233, 04510 México, D.F., Mexico a r t i c l e i n f o a b s t r a c t The interaction between Acacia and Pseudomyrmex is a textbook example of mutualism between ants and plants, nevertheless aspects of its evolutionary biology have not been formally explored. In this paper we analyze primarily the phylogenies of both New World Acacia and of their associated species of ants, and the geographic origin of this mutualism. Until now, there has been no molecular analysis of this relationship in terms of its origin and age. We analyzed three chloroplast markers (matK, psaB-rps14, and trnL-trnF) on a total of 70 taxa of legumes from the subfamily Mimosoideae, and two nuclear regions (long-wavelength rhodopsine and wingless) on a total of 43 taxa of ants from subfamily Pseudomyrmecinae. The monophyly of subgenus Acacia and within the New World lineages that of the myrmecophilous Acacia group was established. In addition, our results supported the monophyly of the genus Pseudomyrmex and of the associated acacia–ants P. ferrugineus group. Using Bayesian methods and calibration data, the estimated divergence times for the groups involved in the mutualism are: 5.44 ± 1.93 My for the myrmecophilous acacias and 4.58 ± 0.82 My for their associated ant species, implying that their relationship originated in Mesoamerica between the late Miocene to the middle Pliocene, with eventual diversification of both groups in Mexico. Ó 2010 Elsevier Inc. All rights reserved. Article history: Received 19 November 2009 Revised 4 March 2010 Accepted 15 March 2010 Available online 20 March 2010 Keywords: Acacia Co-evolution Diversification ages Mutualism Pseudomyrmex 1. Introduction Ecological interactions between species are of great evolutionary relevance, and may be one of the forces that have generated lineage diversification and speciation (Chenuil and McKey, 1996; Stebbins, 1981; Thompson, 1994). The ant–plant symbiotic association is a classic example of mutualism (Rico-Gray and Oliveira, 2007). A high degree of specialization in several of the myrmecophilous plants is manifested in modified housing structures (domatia), and food bodies for the resident ants. In exchange, the ants protect the plant against herbivores, pathogens and/or climbing plants that may obstruct the optimal growth of the plant (Davidson and McKey, 1993; Heil, 2008; Heil and McKey, 2003; Janzen, 1966). Also it has been suggested that there is also production of additional CO2 and nutrients such as nitrogen as a result of the decomposition of debris produced by the ants (Fischer et al., 2003; Sagers et al., 2000). Myrmecophylia has been described in at least 465 species (in 52 families) of vascular plants, including herbs, shrubs, trees and lianas * Corresponding author. Fax: +52 55 56 22 89 95. E-mail addresses:
[email protected],
[email protected] (S. GómezAcevedo). 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.03.018 that maintain this symbiosis with at least 25 ant genera (Jolivet, 1998). This interaction seems to be most common in tropical regions (McKey and Davidson, 1993). It has been reported that in genera with a pantropical distribution, myrmecophylia is only present in either neotropics or palaeotropics, being the genus Acacia the exception (McKey and Davidson, 1993), as it includes four myrmecophilous species in Africa (Janzen, 1974) and 15 species in the American continent (Rico-Arce, 2003). The latter species are also called swollen-thorn acacias. The Neotropical myrmecophilous Acacia species are distributed mainly along the coast and in central areas of Mexico southwards to northwestern Colombia. These species display morphological structures associated with a mutualist ant interaction, such as swollen hollow stipular thorns (domatia), leaflet tips bearing beltian bodies (as food for the ants), and one or more extrafloral nectaries on the leaves (Janzen, 1966). The beltian bodies are a source of food for the ant larvae and contain lipids (1–10% of dry weight), proteins and free amino acids (8–14% of dry weight), carbohydrates (3–11% of dry weight) and water (18–24%) (Heil et al., 2004). All Neotropical species maintain an obligate mutualism with the Neotropical Pseudomyrmex ferrugineus ant group which contains 10 species that have no interaction with any other 1998). 2003. following the phylogeny proposed by Lavin et al. trnL 30 exon. Sequences were generated with four Acacia-based internal sequencing primers from Miller and Bayer (2001). 5 species of subgenus Acacia from the Old World. (2005). 1993).1. Our phylogenetic work is related to the taxonomy more than to the nomenclature. 2009. 2005. we suggest that it is especially relevant to analyze the phylogeny of the groups involved in the interaction. 2003. 1. Johnson and Soltis. PCR reactions followed Taberlet et al. PCR reactions followed Miller and Bayer (2001). 7 species of tribe Ingeae. Maslin et al. Pellmyr. and an intergenic spacer between trnL gene and the trnF gene was amplified and sequenced using primers C (50 -CGA AAT CGG TAG ACG CTA CG-30 ) and F (50 -ATT TGA ACT GGT GAC ACG AG30 ) from Taberlet et al. a genus with three subgenera for several reasons. The final taxa selected included 22 species of subgenus Acacia from the New World (including 10 of the 15 species of the Neotropical swollen-thorn acacias). DNA sequence analysis of Acacia taxa were sampled for each of the main five possible lineages suggested by Maslin et al.. Three cpDNA regions. Dejean et al. Despite the numerous nomenclatural changes suggested for the species in the whole genus. We decided to use Acacia. Pedley..5 lL of 30 mM/L magnesium chloride solution.1. 1994) and an Acacia-based primer Ac1707R (50 -TGC ACA CGG CTT TCC CTA TG-30 . one species of Myrcidris and three species of Tetraponera (from GenBank)... (1991). (2005). The psaB-rps14 spacer was amplified and sequenced using the primers psaB-rps14 1 (50 -GCA CGA TTA GTT GGA TTA GC-30 ) and psaB-rps14 2 (50 -CCA TCT CAC GGA GTA TGT GT-30 ) (Suyama et al. 2. Smith et al. 2006. and some of them suggest that specialist pollination can promote rapid and simultaneous diversification in both the plants and the pollinators (Althoff et al. and in view that nomenclatural issues are pending. which amplified a 2. 10–50 ng of template DNA and 1 U of polymerase in a total volume of 30 lL. we sampled different ant species of the genera Myrcidris. patterns and diversification. 0. 2. Smith et al. ant–plant relationships have been poorly studied (Bronstein. Sampling Four species were collected of living plants. Moore. 13 species of subgenus Aculeiferum (including 4 species of the A. 12 species of subgenus Phyllodineae. For the phylogenetic analysis we used a total of 39 species (35 from GenBank) of Pseudomyrmex – including 8 of the 10 species of the P. or are they dependent on the mutualist association? In addition.2 lL of a 1. Ac283R (50 CAC TGA CGG CAA GCC CCT CTG-30 ).2. The matK gene was amplified using the trnK-3914 primer (50 -GGG GTT GCT AAC TCA ACG G-30 . 2004. Sampling Leaf tissue was either obtained from fresh material (collected in natural populations or sampled from cultivated plants) or herbarium specimens (from the MEXU and USCG herbaria). The P. DNA isolation and amplification Total genomic DNA was isolated from leaves using CTAB protocol (Doyle and Doyle. 2005.5 kb fragment. ferrugineus group. Ac12F (50 -GGT GCA (A/C)AA TCT AGG TTA TGA C-30 ). Mimosoids 2. coulteri group also named Mariosousa). Filicinae). While ecological studies on mutualism are abundant in literature (Bronstein.394 S. P. 2007). (1991). ferrugineus group is distributed from eastern to western Mexico throughout Central America to northern Colombia. For the plants we included members of the tribes Acacieae... all nesting exclusively in Acacia domatia (Ward. Gómez-Acevedo et al. For the ants. 7 species of the genus Acaciella (formerly Aculeiferum sect. 2003. Material and methods A broad sample of the species in both groups. In particular. 1987).. 2. peperi and P. 2004. psaB-rps14 and trnL-trnF. . 2008. Their domatia are occupied by only four ant species: three belong to Crematogaster and one to the genus Tetraponera (Young et al. Whittall and Hodges. P. Orchard and Maslin. we opted to follow the classification of Acacia as one genus with three subgenera. 1993). 55 °C for 1 min and 72 °C for 1 min. 2009). The ants were collected directly from the domatia of Neotropical swollen-thorn acacias. broadly following the distribution of the myrmecophilous Acacia plants. To date the possible retypification of the genus is still inconclusive and unresolved as the inclusion of A. three of them myrmecophylous.. 2006. Gibernau et al. P. 1997). 1994). there is still a controversial issue of which is the most adequate to used based on its pantropical distribution (Walker and Simpson. (2) which are the estimated ages of their lineages?.2. Rijckevorsel. 2008). 2004. Ingeae and Mimoseae (Leguminosae: Mimosoideae). Janzen (1974: p. were amplified via the polymerase chain reaction (PCR). although there is not a single species that covers the whole range (Ward. the vast majority lack analysis of their evolutionary origins. 2008. 2008. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 vascular plant. although the Acacia–Pseudomyrmex interaction is one of the most studied symbioses to date. The African myrmecophilous Acacia present domatia and extra floral nectaries.1. 2000). 2004.. 2003. In particular.. In contrast. 2007..1. matK. (2003) and Lewis et al.. plants and ants. Kato et al. but in contrast to the Neotropical myrmecophilous Acacia they lack beltian bodies (Stapley. Ants 2. penninervis in the printed international nomenclature code will be presented for ratification in the nomenclatural section in the next International Botanical Congress in Melbourne in 2011. with a final extension period of 72 °C for 10 min. The PCR reaction mixture consisted of 3 lL of 10 reaction buffer. was undertaken including selected outgroups. 2008. Ac1290R (50 -AAT ACA AGA AAG CCG AAG-30 ) and Ac1104F (50 -CCT CTA ATT AGA TCA TTG GC-30 ). Grangier et al. Luckow et al. Maslin. 2. Pseudomyrmex and Tetraponera (Formicidae: Pseudomyrmecinae). we discuss the evolution and systematics of the genus Acacia within the Leguminosae and Pseudomyrmex within Formicidae. We used Prosopis as outgroup so we could have a more suitable Mimosoideae taxon as a calibration point..25 mM/L dNTP solution in equimolar ratio. the hypotheses of a rapid and simultaneous diversification have not been tested for the association between the swollen-thorn acacias and its associated ants. and 4 species of tribe Mimoseae (Table 1).1. Amplification was carried out for an initial denaturization of 95 °C for 10 min followed by 40 PCR cycles consisting of 94 °C for 30 s. 2006. 2006. 2003. Most published studies on mutualism cover the interaction between plant and pollinator. The trnL-trnF region.25 mM for each primer. Jousselin et al. 1. and (3) are the diversification rates correlated?. which consists of the trnL intron. or that some non-myrmecophilous species became myrmecophilous by hybridization.2. The following questions were used to guide the study: (1) are both the myrmecophilous Acacia and their mutualists Pseudomyrmex monophyletic groups?. 2000). veneficus and were stored in 70% ethanol. Szilágyi et al. where the mutualism might have evolved more than once. Miller and Bayer. 1994. Murray. mixtecus. Brouat et al. 12) suggested a polyphyletic origin for the American myrmecophilous Acacia species. The aim of this paper is to analyze the Acacia–Pseudomyrmex mutualism from a phylogenic perspective in order to elucidate the evolutionary history of this relationship. 2003. ferrugineus.. constricta A. vogeliana A. globulifera A. The phylogenies of both myrmecophilous group and their associated ants were compared using TreeMap (Page.S. adoxa A. (2006). penninervis A. schaffneri A. The wingless region was amplified using Wg578F (50 -TGC ACN GTG AAR ACY TGC TGG ATG CG-30 . (2003). chiapensis A.2. 1997). Species from Murphy et al. painteri A. caven A. Amplification was carried out for an initial denaturation of 95 °C for 1 min followed by 40 PCR cycles consisting of 95 °C for 30 s. tequilana Tribe Ingeae Albizia kalkora matK psaB-rps14 trnL-trnF Table 1 (continued) Species 61 62 63 64 65 66 67 68 69 70 a b 395 matK HM020736 c psaB-rps14 HM020784 HM020785 b c trnL-trnF HM020834 c 1 2 3 4 5 6 7 8 9 10 11 12 13 HM020708 HM020709 HM020711 HM020713 HM020717 HM020718 HM020719 HM020720 HM020722 HM020729 c c c c c c c c c c HM020745 HM020746 HM020748 HM020750 HM020754 HM020755 HM020756 HM020757 HM020760 HM020769 HM020751 HM020768 b c c c c c c c c c c HM020798 HM020799 HM020801 HM020803 HM020807 HM020808 HM020809 HM020810 HM020813 HM020821 HM020804 HM020820 b c c c c c c c c c c Calliandra houstoniana var..25 mM/L dNTP solution in equimolar ratio. and allowing unlinked parameter estimation among partitions using a Bayesian approach (MrBayes version 3. with a final extension period of 72 °C for 3 min. macracantha A.5 lL of 30 mM/L magnesium chloride solution. pennatula A. For both wingless and longwavelength rhodopsin. collinsii A. Analyses included two paired runs. Species sequenced in this study. Both groups of taxa were analyzed with 5.3. mimosoids and ants. Phylogenetic analyses were conducted implementing gene partitioned data. applying the best fit substitution model. schottii A. 1998) was utilized to find the best-fitting model for the data set for each DNA region of mimosoids and ants separately. bicolor A. (2000). / Molecular Phylogenetics and Evolution 56 (2010) 393–408 Table 1 Mimosoid taxa analyzed in this study. roigii A. mayana A. wingless and long-wavelength rhodopsin. willardiana Subgenus Phyllodineae A. 52 °C for 1 min for wingless/50 °C for 1 min for long-wavelength rhodopsin. were aligned using ClustalW (Thompson et al. farnesiana A. colei A. anegadensis A.0 (Hall. feddeana A. 1994). cedilloi A. were amplified via polymerase chain reaction (PCR). Gómez-Acevedo et al. berlandieri A. sphaerocephala African ant-acacias A.. The program Modeltest (Posada and Crandall. 2005) and Wg1032R (50 -ACY TCG CAG CAC CAR TGG AA-30 . gentlei A.30 min. cochliacantha A. . and subsequently employed for phylogenetic analysis. neovernicosa A. riograndensis A. Species from Miller and Bayer (2003). 1994) as implemented in BioEdit 7.000. angustissima A. 2002). Huelsenbeck and Ronquist.25 mM of each primer. tortilis Subgenus Aculeiferum A.2. hirtipes A. 1. Genbank numbers are given for all cpDNA sequences.2) and a sample frequency of 200 generations. Miller (unpublished). dolichostachya A. scleroxyla A. the PCR reaction mixture consisted of 3 lL of 10 reaction buffer. ampliceps A. gaumeri A. visco A. platycarpa A.2.000 generations. 1996). and 72 °C for 1. Analyzed species of Pseudomyrmex belong to the nine morphological groups proposed by Ward (1989) (Table 2). seyal A. Species from Luckow et al. Species from Seigler et al. Species from J.1. 10–50 ng of template DNA and 1 U of polymerase in a total volume of 30 lL. Ward and Downie.0. drepanolobium A. luederitzii Non myrmecophilous New World A. karroo A. Abouheif and Wray. HM020771 c HM020818 c AF522971 e HM020823 c 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AF274137 AF274140 f f HM020758 HM020770 c c c HM020811 HM020822 c c HM020707 a DQ371892 g HM020716 c DQ371895 g EU812009 h HM020725 c HM020726 c a HM020744 HM020781 HM020782 HM020753 b b c c c c HM020763 HM020764 a b c c DQ371891 g HM020731 c HM020732 c HM020735 c AF523076 e AF523074 e AF274215 e AF274149 e HM020723 c AF274166 e AF274159 f AF274223 e HM020727 c AF523082 e AF274165 f AF523111 e HM020733 a c HM020772 HM020773 HM020783 b b b b c c c HM020797 c HM020832 c DQ371873 g HM020806 c DQ371858 g EU439983 h HM020815 c HM020816 c HM020819 c DQ371864 g HM020824 c a HM020833 AF195684 e AF522983 e AF522987 e AF195683 e AF195694i AF195680 e b c 2. each with one cold chain and three heated chains (temp = 0. cornigera A. usumacintensis A. 0. Phylogenetic analyses Sequences of both data sets. chamelensis A. No sequence found in GenBank. saligna A. melanoxylon A.2. tortuosa Non myrmecophilous Old World A. Species Subgenus Acacia Neotropical ant-acacias A. The long-wavelength rhodopsin was amplified using Lr143F (50 -GAC AAA GTK CCA CCR GAR ATG CT-30 ) and Lr639ER (50 -YTT ACC GRT TCC ATC CRA ACA -30 ) from Ward and Downie (2005). Species from Miller and Bayer (2001). spinescens A. mearnsii A. reniformis A. 0. elata A. 2001). translucens A. Two nuclear regions. centralis A. DNA isolation and amplification Total genomic DNA was isolated from whole ants using phenol– chloroform protocol (Gadau et al. rosei A. picachensis A. HM020761 b b b c HM020766 b b b c AF522985 e HM020817 c AF195687 e AF522984 e AF522986 e HM020825 HM020826 HM020827 HM020828 HM020829 HM020830 HM020831 AF522945 e c c c c c c c HM020734 a a a a c HM020774 HM020775 HM020776 HM020777 HM020778 HM020779 HM020780 b c c c c c c c AF523083 e 2. sousae A. anomala Faidherbia albida Havardia pallens Inga edulis Lysiloma divaricatum Pithecellobium dulce Tribe Mimoseae Mimosa similis Piptadenia viridiflora Prosopis juliflora Prosopis laevigata HM020737 c AF274125 e HM020738 c AF523088e HM020740 c HM020739 c AF521856 e HM020741 c HM020742 c c HM020786 HM020787 HM020789 HM020788 b c c c HM020835 c AF522955 e HM020836 c HM020837 c a c HM020790 HM020791 c c HM020838 c AF522963 e HM020839 c HM020840 c HM020714c HM020728c AF523186d c c c c c d e f g h 14 15 16 17 18 19 20 21 22 23 24 25 HM020706 c AF274131 e HM020710 c HM020712 c HM020715 c HM020721 c AF523113 e HM020724 c a HM020743 b c HM020747 HM020749 HM020752 HM020759 b c c c c AF274132 f AF274136 e HM020730 c HM020762 HM020765 HM020767 b c c c HM020796 c AF522967 e HM020800 c HM020802 c HM020805 c HM020812 c AF522970 e HM020814 c a i Regions that could not been amplified nor sequenced. tumida Genus Acaciella A.8 lL of a 1. hindsii A. 93 1.29 6. 1925).18 ± 1.97 4.00 ± 2.37 18.48 Maximum (My) 139. The letters correspond to those in Figs. 2.92 4. Prosopis with a range between 22.44 ± 1.46 11.29 6.95 8.63 ± 0.38 ± 1.57 ± 2.18 ± 2.79 1.10 7.17 2.81 11.76 Species from Ward and Downie (2005).23 7. Species from Kautz et al.08 9.93 1.6 6. 2002) implemented in r8s version 1.12 ± 0.09 5.33 3.72 2.14 3.48 3.27 13.19 4.68 ± 0.31 ± 2. (2005: p. Node B C D E F G H I J K L L0 M N O P Q R S T U V W Mean age (My) ± SD 18.99 11.76 ± 0.45 32.22 1. discrete-state.18 ± 1. 1 and 2.01 ± 1.05 9.42 ± 2. My = million years.75 ± 0.27 17.51 . 1997.31 ± 2.45 14. n0 is the initial diversity 1.58 6.67 ± 1.52 2.39 17.44 7. based on node 11 given by Lavin et al.66 17.51 3.50 1.4.44 0.80 ± 10.74 Minimum (My) 76.13 9.23 4. We obtained point estimates of age for each node in each of the 72 mimosoid trees topologically identical to the consensus tree and in 103 ant trees topologically identical to the maximum a posteriori (MAP) topology from among those sampled by the Bayesian MCMC’s.37 11.396 S.82 6. 1992).46 11. wg = wingless. My = million years.98 Minimum (My) 17.23 2.83 0.08 3.77 6.88 ± 1. LW Rh = long-wavelength rhodopsin.38 6. For mimosoids.02 ± 0.64 1.97 5.2.92 ± 2.44 5.0 My for the genus Pseudomyrmex was used based on fossil records from Baltic Amber and Dominican Amber fossils. and an age constraint of 15.16 6.92 2. From these.64 ± 1. Gómez-Acevedo et al. For the ants. LW Rh AY703785 AY703786 AY703787 AY703788 AY703789 AY703790 AY703791 AY703792 AY703793 HM020792 AY703794 AY703795 AY703796 AY703797 AY703798 AY703799 AY703800 AY703801 HM020793 AY703802 AY703803 AY703804 AY703805 AY703807 AY703806 AY703808 HM020794 AY703809 FJ436889 AY703810 AY703811 AY703812 FJ436890 AY703813 AY703814 AY703815 AY703816 AY703817 HM020795 AY703818 AY703770 AY703781 AY703783 Wg AY703651 AY703652 AY703653 AY703654 AY703655 AY703656 AY703657 AY703658 AY703659 HM020841 AY703660 AY703661 AY703662 AY703663 AY703664 AY703665 AY703666 AY703667 HM020842 AY703668 AY703669 AY703670 AY703671 AY703673 AY703672 AY703674 HM020843 AY703675 FJ436865 AY703676 AY703677 AY703678 FJ436866 AY703679 AY703680 AY703681 AY703682 AY703683 HM020844 AY703684 AY703636 AY703647 AY703649 Myrcidris epicharisa Pseudomyrmex apachea Pseudomyrmex boopisa Pseudomyrmex concolora Pseudomyrmex cordiaea Pseudomyrmex cubaensisa Pseudomyrmex dendroicusa Pseudomyrmex denticollisa Pseudomyrmex elongatulusa Pseudomyrmex ferrugineusb Pseudomyrmex filiformisa Pseudomyrmex flavicornisa Pseudomyrmex godmania Pseudomyrmex gracilisa Pseudomyrmex haytianusa Pseudomyrmex holmgrenia Pseudomyrmex itaa Pseudomyrmex kuenckelia Pseudomyrmex mixtecusb Pseudomyrmex nigrocinctusa Pseudomyrmex nigropilosusa Pseudomyrmex oculatusa Pseudomyrmex okia Pseudomyrmex pallensa Pseudomyrmex pallidusa Pseudomyrmex pazosia Pseudomyrmex peperib Pseudomyrmex phyllophilusa Pseudomyrmex satanicusc Pseudomyrmex sericeusa Pseudomyrmex simplexa Pseudomyrmex spiculusa Pseudomyrmex spinicolac Pseudomyrmex subatera Pseudomyrmex tachigaliaea Pseudomyrmex tenuisa Pseudomyrmex tenuissimusa Pseudomyrmex termitariusa Pseudomyrmex veneficusb Pseudomyrmex viduusa Tetraponera aethiopsa Tetraponera pilosaa Tetraponera rufonigraa Table 3 Age estimates (means and standard deviations) derived from 72 Bayesian trees sampled similar to the consensus tree for legumes. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 Table 2 Ants analyzed in this study.00 1. Ward.82 4.96 10. under a pure birth model using the formula SR = (lnn1 À lnn0)/t.90 ± 0.34 7.10 1. Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 a b c identical trees to the consensus tree due that we only obtained three trees topologically identical to the MAP topology.09 10.65 6.57 ± 1. For the mimosoids. we used the 72 Table 4 Age estimates (means and standard deviations) derived from 103 Bayesian trees sampled similar to the tree of major posterior probability for ants.42 ± 1.75 ± 1. This latter node age is based on the node G of Lavin et al.93 ± 2. and t is time in My (Yule.85 4.5.89 6.85 ± 2.25 7.66 ± 2.0–20. Markov process in which the number of splitting events along any path in a tree follows a Poisson distribution (Sanderson and Wojciechoswki.78 ± 5.80 9.2. (2005: p. The letters correspond to those in Figs. Diversification rates Diversification rates were estimated only for the monophyletic groups of both mimosoids and ants.99 9. 1996). this method provides a good first approximation for diversification estimates (Sanderson and Donoghue. Species sequenced in this study.97 2.82 40.49 4. (2009).98 10.35 1. a mean and standard deviation of the age of each node were obtained (Tables 3 and 4). It assumes an effectively constant rate of lineage diversification with no extinction. 1994).35 1.4 My was used.31 5.29 10.55 5.58 ± 1.16 2. 581) and a minimal age of 15.38 ± 1.74 17.02 19.65 4.83 2.35 14.69 ± 1.67 9.63 5. 2003) using the consensus tree for mimosoids and the maximum a posteriori (MAP) tree for ants. where n1 is the estimated number of extant species.48 ± 1. SD = standard deviation.63 4. 2.56 8.32 8. 581).07 4.67 8.51 12.6 and 40.24 2.56 2.73 2.58 ± 0.30 Maximum (My) 19.08 3.14 5. The Yule model is a continuous time.02 12.63 14. Two calibration points were used in the analysis for both data sets.93 ± 1.97 ± 3.0 My for the clade that includes all the species here sampled except Prosopis sp. respectively (Dlussky.34 ± 2.54 1.85 9.33 ± 1.64 1. In spite of its simplicity. SD = standard deviation.11 12.98 16.16 17.08 ± 3.07 3.68 ± 1.90 12. Species numbers (n1) were based on a review of the literature.96 4.22 11. Divergence times Divergence times were estimated using penalized likelihood (Sanderson.27 1.19 6. an age of 44.32 13.71 (Sanderson.34 3.36 12.60 6.72 3.86 13.52 1. GenBank numbers are given for all nDNA sequences.10 My for genus Tetraponera.19 6.87 0. 3 and 4.41 14. Node A0 B D E F G H I J K L M N O P Q R S T Mean age (My) ± SD 95.23 3.06 ± 2.52 17.8 54.56 0. Acaciella chamelensis nested separately with six of the seven species of Ingeae. Inga edulis.33 ± 1. 2008). Figs. macracantha group (clade K. The Caribbean endemic species A. willardiana diverged before node U. 2006). Phyllodineae. dolichostachya. Clade V includes two endemic species from Bolivia (A. laevigata were selected as outgroup taxa (clade A. usumacintensis and A.4. A. was found to be non-monophyletic be- cause A. A.321 sp/My for all subgenus Acacia from the New World (53 species) and 0. Aculeiferum. including branch lengths. including Acacia subgenus Phyllodineae. Lewis et al. with a rate . Faidherbia albida. Genus Acaciella The seven studied species of this genus belong to two clades. Within this group it is possible to distinguish a small clade formed by A. although it was represented by only 12 species – a small sample size considering its estimated size of 1045 species (Miller and Bayer. collinsii. tortilis. Acaciella is established as paraphyletic.02 My (clade N. are shown in Fig.82 My (Figs. using estimates of the total number of extant species of groups. including six species of the sampled Ingeae. 3.97 ± 3. as suggested for Yucca (see Smith et al. These species are grouped with A.424 sp/My for its sister group. represented from either fresh or herbarium material. 1 and 2 and Table 3). acuifera group. anomala as their closest relative (clade P). 1 and 2). 3. A. 1 and 2). Six of them. scleroxyla) (Figs. 1 and 2). Subgenus Aculeiferum The analyzed species of subgenus Aculeiferum were non-monophyletic. the A. species of the subg. drepanolobium and A. respectively (Figs. coulteri group (A. The dated phylogeny is shown in Fig. willardiana) within the clade U (Figs. This clade has an estimated age of 6. 1 and 2).66 ± 2. seyal and the non-myrmecophilous A. farnesiana. or Mariosousa). Lysiloma divaricatum. Acacia constricta. centralis. berlandieri. 1 and 2).S. A.6. sister to the non-myrmecophylous A. subgenus Phyllodineae. which belonged to two clades. clade L. A. The Neotropical swollen-thorn acacias are monophyletic (clade L. Tribe Ingeae Tribe Ingeae was represented by seven genera. cochliacantha. coulteri group. 2..32 My (Figs. In contrast. Clade W contains A. The A. A. New and Old World species grouped into two different clades.1. neovernicosa and A.1..8. and the other grouping species of subgenus Aculeiferum. coulteri group. Gómez-Acevedo et al. The species A. In general. 0. luederitzii (E in Figs. These Acaciella species had an estimated age of 4. Fig. tribe Ingeae as well as the species of the genus Acaciella. The only troublesome sequences were those samples obtained from herbarium specimens of Acaciella. globulifera. 3.5. 1 and 2). and species of tribe Ingeae and species of tribe Mimoseae. The ages of these groups are listed in Table 3. hirtipes (clade L0 in Figs. G and D. Figs. A.496 sp/My (15 species) in contrast to the 0. but all belong to a clade dated at Miocene (clade O in Figs.1. A. The first includes A. In total we used a matrix of 70 taxa from which we amplified and sequenced 37 species for matK.07 My (Figs.1. 1 and 2). 1 and 2).3. The estimated ages for these groups are listed in Table 3. 1 and 2). 1 and 2). 1 and 2 and Table 3).67 ± 1. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 397 3. The ages of these groups are listed in Table 3.341 sp/My (considering a total of 161 species). Calliandra houstoniana var. K81+uf+G for psaB-rps14 and TIM+G for trnL-trnF the data sets of mimosoids. 3. 1 and 2 and Table 3). Its crown age was estimated as 3. karroo (F in Figs. Mimosa similis and Piptadenia viridiflora were grouped together as a basally branching sub-clade of 9. picachensis (Figs. most groups are supported by high posterior probabilities (Fig.2. we estimated the diversification rates of monophyletic groups within the studied mimosoids. 1). roigii were established in clade M (Figs.395 sp/My for its sister group from the Old World (73 species). visco is the sister group of a clade that includes all sampled species of the subg. whereas Prosopis juliflora and P. 1 and 2). As mentioned above. Havardia pallens. 1 and 2). 1 and 2). 3. Figs. A. riograndensis) and one from the Dominican Republic (A. tortuosa are nested in clade I (Figs. one grouping all species of subgenus Acacia. 2003. 3. thus. hindsii and A. caven. 1 and 2). recently proposed as the genus Mariosousa (Seigler et al. reniformis. A. The models with best fit to the sequences were GTR+I+G for matK. Albizia kalkora. Subgenus Phyllodineae Subgenus Phyllodineae was resolved as monophyletic. Diversification rates in the mimosoids In order to test the idea that the specialized mutualism between the ants and Acacia may have increased the diversification rates of both mutualistic species. pennatula were found in clade K (Figs. gaumeri and A. including also Acaciella chamelensis and the 12 species of subgenus Phyllodineae (Figs. Six of them form a monophyletic group (clade Q) with Calliandra houstoniana var. 1. Estimated ages for these groups are listed in Table 3. Tribe Mimoseae Tribe Mimoseae was represented by four species. and those of the tribe Ingeae and genus Acaciella. Considering the informal groups from the New World. The DNA sequences obtained from GenBank and used in these analyses are indicated in Table 1. Phyllodineae.7. Mimosoids Amplification and sequencing of the three chloroplast regions was achieved relatively ease for all species. macracantha and A. the known African swollen-thorn acacias were not established as monophyletic but instead nested into two sub-clades of the clade D. Results 3. 1 and 2).. A. 1).1. 3. 49 species for psaB-rps14 and 45 species for trnL-trnF. A second sub-clade grouped the myrmecophilous A. with a rate of 0. the segregate genus Acaciella. macracantha group (7 species). Phylogeny and divergence times Phylogenies estimated for the set of 70 taxa using the concatenated matrix of 3128 sites with a Bayesian approach. 1 and 2). 2005). corresponding to clade S. chamelensis). The seventh species of Ingeae. the prediction of high diversification rate due to the specialized mutualism holds. anomala was nested together with six species of Acaciella (P in Figs. 1 and 2 and Table 3). schaffneri and A. and a single species of Acaciella (A. All taxa analyzed were included in one of two major clades. schottii diverged before to the rest of the New World species in clade H (Figs. A.1.1. 1 and 2) and are closely related to the A.1. The total rate for subgenus Acacia was 0. The second highest rate was obtained by the A. as the highest rate was found in the myrmecophilous group. Subgenus Acacia This subgenus was resolved as a monophyletic group with the origin of the crown group dated at Miocene (labeled as C in Figs. A.1. feddeana and A. 3. as the sister group of this clade that includes species in the subgenera Aculeiferum (including the four species of the A. anegadensis and A. and Pithecellobium dulce clustered in clade R. cedilloi.1. These species are nested within clade R. vogeliana and the four species belonging to the A. macracantha. Gómez-Acevedo et al.457 sp/My (12 species). The diversification rate of the Neotropical myrmecophilous. A. Thicker horizontal lines represent branches supported by posterior probabilities of 0. and A. constricta group has the lowest rate among the groups of the New World. The letters indicate the groups discussed in the text. The A. of 0.398 S. 1. inferred from the three chloroplast data (matK.201 sp/ My (11 species) (Table 5). Bayesian consensus tree of Acacia and selected relatives. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 Fig.50. its rate is of 0. acuifera groups are .00. psaB-rps14 and trnL-trnF).90–1. Dashed lines represent branches supported by posterior probabilities of equal or above 0. (2005). Dashed lines represent branches supported by posterior probabilities of equal or above 0.90– 1. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 399 Fig. Chronogram derived from the all-compatible Bayesian consensus tree of Acacia and selected relatives. a = 0. P(d.087 sp/My . higher than the rates of the other New World groups (F = 30.50.0000).S. as derived from Lavin et al. Thicker horizontal lines represent branches supported by posterior probabilities of 0. No branches below 0.4 My. = 4. 2.f.00.05) = 0. Gómez-Acevedo et al. The root was considered at range between 22. The letters indicate the groups discussed in the text.50 were resolved in the Bayesian consensus. It is important to remark that subgenus Phyllodineae has the highest diversification rate of all sampled groups.6 and 40. 2.72. this is a monophyletic group supported by a high posterior probability of 0. F. (1045 species). Figs.502 sp/My. ferrugineus (clade N).68 ± 1.2. The time of . and 0.90 and an estimated age of 4.341 0. Conversely. Pseudomyrmex The data resolved a monophyletic Pseudomyrmex (clade C) with an estimated age of 20. is monophyletic and is the sister lineage to Myrcidris plus Pseudomyrmex (clade A. it is important to note that swollen-thorn acacias with major geographical distributions such as A. ferrugineus and P. the groups of P. Nevertheless.82 My.264 sp/My. the diversification rates are similar. ferrugineus group and P. In general. P.34 ± 2. Also. ferrugineus group sampled were collected fresh. From the nine morphological groups suggested by Ward (1989). The ages for these groups are listed in Table 4. The beltian bodies contain 10 amino- a Acaciella comprises 15 species. a species endemic to Haiti. chamelensis grouped within the tribe Ingeae) has an intermediate rate of 0. The reciprocal adaptations between myrmecophilous acacias and its mutualistic ants reinforce the co-evolutionary proposal. cornigera and A.563 2. 3 and 4 and Table 4). The number of species is the total for each group.93 My.321 sp/My. pallidus group with 1. tenuis group (Fig. The highest rate was for the P. ferrugineus. Fig. due to the fact that these ants nest exclusively in the domatia of myrmecophilous acacias.264 sp/My (200 species). 1) was 5. Myrcidris and Tetraponera Myrcidris epicharis was found to be an early diverging branch.58 ± 0. The diversification rates of the seven monophyletic groups are shown in Table 5.321 0. P. the Pseudomyrmex pallens group (clades G. collinsii. Also. Fig. Phylogeny and divergence times The phylogeny estimated for the set of 43 taxa.82 My (clade N. 4. Figs. Figs. tenuis group with 0. P.88 ± 1. the diversification rate was obtained from the monophyletic clade of six species. and that each species of Acacia could be exploited by several Pseudomyrmex ants. Q. we analyzed eight of ten species of the P.496 sp/My for the ant–acacias. Figs. Discussion 4.400 S. such as P. haytianus. 3 and 4 and Table 4). 3.51 My (clade B. P.502 1. On the contrary. represented by three species T. the origin of the myrmecophilous specialized Acacia (clade L. 1) have an age of 12. but for the analysis only 14 species were considered.58 ± 0. T) were non-monophyletic. pallidus (clade P).913 0. 2005). 3).1.2.502 sp/My for the acacia–ants. 3. 3 and 4 and Table 4). 3 and 4) and with two primary lineages (clades D.410 0..3. derived from our data and data from the literature. species with restricted distributions in both taxa have fewer associate species. amplified and sequenced without any problem for two nuclear regions.80 ± 10. hindsii are inhabited by a large number of P. (clade C. including branch lengths is shown in Fig. 39 of them were obtained from GenBank (Table 2). The Pseudomyrmex genus with a similar age. Six major clades are nested in clade F with an estimated age of 17. Tetraponera. in general has a slightly lower diversification rate. sericeus (clade J). which is sister to genus Pseudomyrmex. 3) is similar. 3.82 My (clade A0 .496 0.147 sp/ My (4 species). The divergence age for the New and Old World lineages was 95. the acacia–ants that have a greater distribution are associated to many swollen-thorn acacias.911 divergence between the P. pallidus and P. 2004. the New World species belonging to this subgenus (clade G. there is not a one to one pattern of specialization.984 0. Acacia–Pseudomyrmex mutualism The Acacia–Pseudomyrmex symbiosis has been often cited as an example of a co-evolutionary relationship since Janzen (1966).0 My (Figs. peperi.201 0. except for the node shared by the P. The sister group of this clade is P. 3. The tree with estimated ages is shown in Fig. using a concatenated matrix of 980 sites and a Bayesian approach.2.2. rufonigra. Diversification rates in Pseudomyrmex The diversification rates for the sampled species was estimated as 0. aethiops.563 sp/My (Table 5).147 0. in which the specialist species (either of acacia or ants) mainly interact with a proper subset of those species interacting with the more generalist species..1. It is clear that each ant species could be associated to several Acacia species. 0. 0. ferrugineus group ants. pilosa and T.23 My (Figs.5.264 0. since it proved to be a non-monophyletic group (see Figs.395 0.136 0.4. whereas the Acaciella genus. The age of this divergence is estimated as 40. 3 and 4 and Table 4).2.18 ± 1. P. 1 and 2) and thus. and has an estimated diversification rate of 0. L) and P. subtilissimus (clade H) and P. 3. haytianus is 6. whereas the lowest rate was found for the P. The mutualistic swollen-thorn acacia ants of the P. had an intermediate diversification rate of 0. ferrugineus group. as we show in Fig. tenuis (clade E) were monophyletic (Figs. Ants The four species of the P. 4.457 0.378 0. These patterns correspond in general terms to a ‘‘highly nested matrix” of mutualistic networks (sensu Bascompte et al. 3 and 4 and Table 4). oculatus (clade S). viduus (in part) groups and that of the P. ferrugineus group with a total of 10 species. In particular. Gómez-Acevedo et al. for which we consider only 14 species (since A. T.98 My. Acacia and Pseudomyrmex dates of origin Subgenus Acacia. 4 and Table 4).087 0.341 sp/My. P. Figs. while our estimate for the clade of the associated ants (N. 5. the chemical composition of the beltian bodies and extra floral nectar are specific for the resident ants (Heil et al. 3.07 My (clade M. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 Table 5 Diversification rates of groups of both mimosoids and ants. 2003). Fig. 3 and 4). Fig.2.78 ± 5.25 My and a diversification rate of 0. In this sense.136 sp/My (27 species). posterior probabilities associated to clades are high. Whereas the diversification rate of myrmecophilous Acacia is the highest within subgenus Acacia. Conversely.2. gracilis (clade I). and the second is the number of species included (in parenthesis). Clade Mimosoids C Subgenus Acacia D Subgenus Acacia Old World G Subgenus Acacia New World H Acacia constricta group I Acacia farnesiana group K Acacia macracantha group L Myrmecophilous group M Acacia acuifera group Q Genus Acaciella S Subgenus Phyllodineae Ants C Genus Pseudomyrmex E Pseudomyrmex tenuis group H Pseudomyrmex subtilissimus group I Pseudomyrmex gracilis group J Pseudomyrmex sericeus group N Pseudomyrmex ferrugineus group P Pseudomyrmex pallidus group S Pseudomyrmex oculatus group Number of species 161 (27) 73 (5) 53 (22) 5 (3) 11 (4) 7 (3) 15 (10) 12 (2) 14a (6) 1045 (12) 200 (39) 4 (4) 4 (2) 31 (3) 15 (3) 10 (8) 27 (4) 11 (2) Diversification rates 0. 3. Fig. We used a matrix of 43 taxa. The models with best fit to the data were K80+G for wingless and TVM+G for long-wavelength rhodopsine.44 ± 1. viduus group (clades K. A. the diversification rate of ants associated with the Acacia is moderate compared to the rest of the Pseudomyrmex genus (Table 5).424 0. Particularly. 4. 1) was originated 14. 3 and 4). Likewise. As stated above. oki. as well as on the presence of Pseudomyrmex in Dominican amber (the result of a radiation from Mesoamerica to the Antilles. an endemic species from Haiti. 2007). 2004). Also. acids and oily acids. 1992). which are essential for the growth and normal development of insects in general (Heil et al.82 My) support this co-evolution proposal and provide an estimate of the time of origin of the mutualistic relationship among Acacia and Pseudomyrmex. At the macroevolution level. the lack of sucrose in extrafloral nectar confers an advantage to the resident ants which lack invertasa. mostly along both coasts up to the Tropic of Cancer (Janzen. 1988). like linoleic and linolelic acids. 4). filiformis have not currently been assigned to any informal group. The letters indicate the groups discussed in the text.. ferrugineus group and P. Gómez-Acevedo et al. In contrast. he suggested that mutualism might have evolved more than once.. ferrugineus group (4.00. with an eventual diversification northwards into Mexico. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 401 Fig. the ages of divergence obtained here for both the Neotropical myrmecophilous acacias (5.90–1.58 ± 0. haytianus and P. haytianus. Kautz et al. 2. ferrugineus group as well as the myrmecophilous Acacia resides in Mexico. 1969). 12) as mentioned above suggested a polyphyletic origin of the American myrmecophilous Acacia species. Rico-Arce. Specifically. The phylogenetic relationship between the P.60. Dashed lines represent branches supported by posterior probabilities of equal or above 0. suggests a historical connection between the Greater Antilles and Mesoamerica. our results suggest that Mesoamerica is the centre of diversification of the mutualistic association between Pseudomyrmex and Acacia. Ward. Janzen (1974: p. the enzyme responsible of sucrose hydrolysis (Heil et al.44 ± 1. clade N in Fig. the present study finds . 1966. P. The highest diversity of species in both P. This expansion was perhaps assisted by bird dispersal (Janzen. 2004. or that some non-myrmecophilous species became myrmecophilous by hybridization. P. Pseudomyrmex subater. Bayesian tree of maximum a posteriori (MAP) topology of Pseudomyrmecinae ants inferred from the two nuclear data (long-wavelength rhodopsine and wingless). This biogeo- graphical pattern has been previously reported for some Caribbean insects (Liebherr. This mutualistic relationship could be situated between the late Miocene and the early Pliocene (clade L in Fig.93 My) and the P. 1966. 2009).S.. 3. Thicker lines represent branches supported by posterior probabilities of 0. Based on this evidence. 2003). sphaerocephala grow also in seasonally dry forests. Chronogram derived from the all-compatible maximum a posterior (MAP) tree of Pseudomyrmecinae ants. 1 and 2 and Table 3). hindsii and A. P. The letters indicate the groups discussed in the text. luederitzii and A. A.90–1. 1 and 2 and Table 3). with A. It is worth mentioning that the Neotropical myrmecophilous acacias and the A. drepanolobium. A. which are produced by plants inhabiting mesic forests. A.3 1. macracantha groups are closely related by common ancestry dated at 6. the acacias are associated with the Brachymyrmex obscurior (Formicidae). However. A. Thicker lines represent branches supported by posterior probabilities of 0.49 My (J in Figs.8 E Pseudomyrmex viduus Pseudomyrmex dendroicus Pseudomyrmex oculatus Pseudomyrmex cubaensis Pseudomyrmex subater Pseudomyrmex tachigaliae Pseudomyrmex concolor Pseudomyrmex simplex Pseudomyrmex pazosi Pseudomyrmex pallidus Pseudomyrmex holmgreni Pseudomyrmex oki Pseudomyrmex nigrocinctus Pseudomyrmex flavicornis Pseudomyrmex ferrugineus Pseudomyrmex mixtecus Pseudomyrmex peperi Pseudomyrmex veneficus Pseudomyrmex satanicus Pseudomyrmex spinicola Pseudomyrmex haytianus Pseudomyrmex phyllophilus Pseudomyrmex pallens Pseudomyrmex kuenckeli Pseudomyrmex sericeus Pseudomyrmex ita Pseudomyrmex cordiae Pseudomyrmex nigropilosus Pseudomyrmex gracilis Pseudomyrmex godmani Pseudomyrmex tenuissimus Pseudomyrmex spiculus Pseudomyrmex elongatulus Pseudomyrmex apache Pseudomyrmex termitarius Pseudomyrmex denticollis Pseudomyrmex tenuis Pseudomyrmex boopis Pseudomyrmex filiformis Myrcidris epicharis Tetraponera aethiops Tetraponera rufonigra Paleocene Oligocene Miocene Pliocene Fig. although A. At this time. With regard to the relationships of the African myrmecophilous species. in this case the ants obtained their food from the extrafloral nectar but they did not protect the plants from the herbivorous (Moya-Raygoza. Their preferred habitat is mesic forests. Pseudomyrmex subater. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 T R S Q P O N M L K F J I C H G B D A ’ A 65.92 ± 2.402 S.60–0. 2005). as derived from Dlussky (1997).00. 1 and 2 and Table 3). hirtipes (L0 in Figs. which lack any distinct morphological synapomorphies. (2000). Additionally. whereas dashed lines represent branches by posterior probabilities of 0. with a diversification age corresponding to the late Miocene (L in Figs. collinsii. A. pennatula (A. Gómez-Acevedo et al. chiapensis. it is important to note that A. P. hindsii being the most widely distributed species of the group (Rico-Arce. 4. globulifera have globose capitate inflorescences. all sampled Neotropical myrmecophilous species forming a monophyletic group. while the rest have spicate inflorescences. collinsii. macracantha group) secretes extrafloral nectar only at the end of the dry season.8 5. this may be related more with the diversification and presence of numerous types of extrafloral nectaries shared by all myrmecophilous species. hindsii and A. The root was fixed at 44. oki. A. Of the ten analyzed Neotropical myrmecophilous species. globulifera.80. In our opinion. haytianus and P.0 Cretaceous 54. Another possible morphological synapomorphy are the modified floral bracts which are peltate in the American myrmecophilous group.7 23. seyal were not estab- . As suggested by Clarke et al. cedilloi. chiapensis and A. A. this is supported by the presence of large leaves that emerge from long shoots. filiformis have not currently been assigned to any informal group.1 My. and the species share no habitat preference. A.8 Eocene 33. The clade includes a subclade formed by A. Kay et al. the highest diversification rates correspond to the A. which suggests that myrmecophylia in Africa occurred slightly earlier than in the Neotropics (E. Janson and Davies. usually at low altitude. 2003. 1999. In contrast. On the other hand. farnesiana and A. Table 5). Could it be that the latter species represents the ancestral radiation of myrmecophylia in Africa? A. 0. 1998) and the essentially Mexican genus Agave (Agavaceae) (0.51 ± 0. as well as for the New World clade (0.. For the A.77–1. calculated from data in Richardson et al. for example. For Acacia hirtipes there is no information of associated mutualist ants. Likewise. seyal myrmecophylous and in the non-myrmecophylous A. respectively.54 sp/My.378 sp/My and 0.44 and 7.. macracantha groups diversification rates are slightly higher (0. but in particular for mutualistic systems..395 sp/My. 2004. whereas in the clade of the Old World species the rate of diversification increases (0. that is. 2002). Cardillo et al. floral peltate bracts are present both in A. Table 5). Table 5) which is distributed mainly in dry environments.. Klack et al. but it was not sampled in this study.2. ferrugineus group. This scenario is similar to that found for other ant–plant mutualisms like in Azteca (Formicidae)-Ceropia (Urticaceae) (Ayala et al. constricta group (0. Marshall et al...424 sp/My. Table 5). host changes are more probable than cospeciation because they involved species that commonly differ with the species which with they interact (Brouat et al. Diversification rates Rates of diversification have been recently used to understand the evolution of biological diversity. the lowest diversification rate corresponds to the A. Davidson and McKey. allowing the identification of groups with exceptional high diversification rates.201 sp/My. Table 5). the Cicadaceae genus Kikihia (0. 2008). Table 5).00 ± 2. Nevertheless. it is generally accepted that reciprocal selection results in congruent phylogenies between insects and plants (Johnson and Clayton. 5). Baldwin and Sanderson. the Hawaiian silversword (Asteraceae) (0. and recently groups of ants have been observed on their thorns (Lubertazzi. Weiblen and Bush. 1 and 2 and Table 3). 2005..17 sp/My. 2004.321 sp/My. respectively.5 sp/My. both comprising species distributed mainly in wet tropical regions. 2008). karroo. we found that the diversification rates increase as the distribution of the species approaches to the wet tropics.563 sp/ My.06 sp/My. The lines between trees indicate the associations reported until now in literature. 1996).6 and 2. A better understanding concerning the latter is still pending. For the Neotropical myrmecophilous acacias. Table 5) which are similar to those obtained for species of the Old World.72 sp/My. The trend of finding the highest diversification rates in the wet tropics has also been reported for Angiosperms in general as well as for animals such as the passerine birds and butterflies (Cardillo. The African species do not have the great diversity and abundance of extrafloral nectaries as found in the Neotropical species. suggesting that myrmecophylia was derived or lost at least twice in the Old World. the diversification rate obtained here for the genus Acaciella is high (0. Malcomber (2002) suggested that in the genus Gaertnera (Rubiaceae) the high diversification rate (0. In contrast. are the result of recent radiations (Hughes and Eastwood..832 sp/My) is correlated with a change in corolla morphology.56 ± 0.496 sp/My. which is probably the consequence of a recurrent change of ant hosts. 5. 2001a). 2004).. 2005). acuifera (0. for the Neotropical genera Costus (Costaceae) and Lupinus (Leguminosae) with values of diversification rates. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 403 A chiapensis A mayana P nigrocinctus P veneficus A gentlei P peperi A cornigera A sphaerocephala A cedilloi A hindsii P ferrugineus A hirtipes A collinsii A globulifera P satanicus P mixtecus P flavicornis P spinicola Fig. which . the diversification rate for the whole subgenus Acacia is relatively low (0. depending on its geographic availability (Fig. Quek et al. 2004) and Petalomyrmex (Formicinae)-Leonardoxa (Leguminosae) (Chenuil and McKey. The divergence ages for the nodes in which these species were nested are 5. Congruent with these. Tanglegram showing relationships between the swollen-thorn acacias and the Pseudomyrmex ferrugineus group. 1996).341 sp/My. 2006). Within the groups in subgenus Acacia. 1993). the co-evolution of these systems probably follows a geographical mosaic in which the populations differ in their characteristics and specializations with respect to the species with which they interact (Thompson. Crematogaster (Myrmicinae)-Macaranga (Euphorbiaceae) (Feldhaar et al. both Neotropical genera of recent origin. lished as sister species. Gómez-Acevedo et al. Pseudomyrmex satanicus is considered the only inhabitant of Acacia melanoceras (Neotropical ant–acacias group). and the Neotropical myrmecophilous groups (0. Good-Avila et al. 2002). 2005).. However.6–2. the South African Aizoaceae (0. karroo has great variation on stipular thorns. 2006.11 My.457 sp/My.717–0.75 sp/My.S. Table 5) and similar to the one of the genus Inga (Leguminosae) (0.08 ± 3. respectively. The comparison of the phylogenies obtained for the Neotropical myrmecophilous acacias and its mutualistic ants reveals the absence of a phylogenetic congruence. its high value might be also due to the mutualistic association with the ants of the P. based on their Bayesian trees. F in Figs. 4.49–3. Our results confirm that tribes Acacieae and Ingeae as traditionally circumscribed are not monophyletic (Figs. 1 and 2). Nevertheless. Congruent with this.. Table 5). Lauraceae and Polygonaceae. 1991). Moreau (2008) assessed a divergence age of 58.136 sp/ My. macracantha (Seigler and Ebinger. acuifera (Clarke and Seigler. pollen grains grouped into 16–32-grained polyads with a columellate exine. The phylogenetic position of the A. Several authors have attempted to classify species from both lineages into different morphological groups. Figs. constricta and A. Luckow et al. Phyllodineae. 1990). only two species of the A. 2008). 1 and 2 and Table 3). A. It seems likely that all these characters were probably acquired only once during the evolution of this taxon. and all were supported as monophyletic (Figs. The monophyly of this subgenus is supported by the following morphological features: stipular spines. .108–0.88 ± 1. we consider Acacia as a single genus comprising three subgenera. 2000). Systematics and phylogeny of Acacia The tribes Acacieae. and in consequence. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 may have induce adaptative morphological features such as proliferation of extrafloral nectaries on the leaves and the unique presence of high-energetic beltian bodies at the tip of leaflets. principally in plants of Leguminosae. gracilis group (0. Chappill and Maslin. G. 2000. 1995. but not scattered prickles. Robinson and Harris. the diversification rates of insects vary from 0. P. Four species representing the A.34 ± 2. In our study. 2009). Table 5) has a low diversification rate. and the myrmecophilous Acacia species (Janzen.. A generally accepted hypothesis considers that Acacieae and Ingeae were derived with respect to the Mimoseae.087 sp/My (Table 5). 2008.. respectively (clades D.. although the species of the P. Miller and Bayer.147 sp/My. sericeus group (0. A. 2001). sericeus group have been related with plants of other families such as Boraginaceae. Our analyses supports the monophyly of subgenus Acacia and suggest a time of origin at 14.410 sp/My and 0. 2003). oculatus group (0. only the P. 2003). 2003.. with a diversification rate of 0. subtilissimus and P. and Yucca (Agavaceae) with 0. Phylica (Rhamnaceae) with 0. Figs. Subgenus Acacia comprises 161 species with pantropical distribution. 2008.33 ± 0.25 My.264 p/My (Table 5). previous molecular studies have shown that this subgenus is monophyletic (Brown et al. Acacia sensu lato flowers have numerous free stamens. In contrast. pallidus group (1. Subgenus Phyllodineae presents the highest diversification rate found in this study. the Pseudomyrmex genus is characterized by arboreal ants. 1 and 2)..913 sp/My. This subgenus comprises 1045 species distributed mainly in Australia and it has been suggested that this high species number could be the outcome of aridification occuring in that continent 10–7 My (Bowman and Yeates. Table 5) have very high diversification rates. all supported by small morphological differences: A. in earlier analyses its members have been also resolved as monophyletic (Clarke et al.18 sp/My (Verboom et al.. constricta. 1989). corresponding to 0.87–4. choriophylla (Clarke et al. These have offer new habitats for colonization. the A. The P. 2003. 2009). 1988). A. Although more recent analyses in which African species were included and nuclear markers were used. farnesiana group has proved to be non-monophyletic (Brown et al. Miller and Bayer. A. This is congruent with Miller and Bayer (2001. 1991. However. which are the predilect habitats of these groups of species. a period in which arid environments increased as a result of global warming (Zachos et al. Our results support this hypothesis and establish two well resolved clades: one containing the New World species and the other the Old World species. 2001.404 S.. ferrugineus group is in part due to its obligatory mutualism with the Neotropical myrmecophilous acacias. Moreover. 2003). Based on this. This group traditionally comprises 11 species. species in the New World lineage have been placed into seven different informal groups.3. anegadensis and A. Clarke et al.. 4. This is supported by the existence of related fossils dated from the middle Miocene to the Pliocene (Graham. Figs. 1 and 2 and Table 3). farnesiana group are monophyletic (clade I.. subg. 2003. the most speciose ant genus (1100 species). Conceivably this lower diversification rate in the P. 2003). 2006). and subg. Following this proposal.626 sp/My (calculated from data in Richardson et al. 2000. tenuis group (0.. the lowest found so far for this genus. the highest diversification rates obtained in this study might be due to the association of these groups of ants with a broader number of plant species. 2001. I. P.. 2003. 1991. acuifera. 1 and 2 and Table 3) which includes 12 species.85 ± 2.06 sp/My (Good-Avila et al. 1 and 2 and Table 3).17 sp/My in Laupala (Gryllidae). higher than the one reported for Pheidole. Figs. The A. for Pheidole (Formicidae). Miller and Bayer. 1989). 2001). 2005). A.. Similarly to the diversification rates of Agave (Agavaceae) with 0.. 1978. 2005). ferrugineus groups have lower diversification rates than the latter groups. all correlated with this aridification process. 1989). Aculeiferum. Robinson and Harris. overall. contrasting with those taxa of tribe Mimoseae which present ten or less free stamens per flower..06 sp/My (Smith et al. A. 2005). A.51 ± 0. constricta group was resolved as the basally branching lineage within the American clade (H in Figs. In the present analysis species of five of these seven informal groups were studied (A. acuifera group were analyzed in this study (A. 2001b). It has been suggested that the New World species of subgenus Acacia resulted from a separate radiation from that of Old World species (Guinet and Vassal. Table 5) and the P. Table 5). Miller and Bayer. in which many species nest in branches or dead leaves among different plant families.. rigidula (Lee et al. A. P.103 sp/My. Although groupings of Old World species have not been convincing (Ross. being 2. physiological adaptations such as the secretion of nectar without sucrose by these nectaries have been suggested as an adaptation that prevents not useful mutualistic visitors to the plant (Heil et al.048 to 0. whereas members of the tribe Ingeae are traditionally defined by its flowers with numerous stamens united into a tube. 1 and 2 and Table 3). 1974). its highest diversification rate could be the result of speciation in response to aridification. we consider that the obligatory Neotropical mutualism Acacia–Pseudomyrmex has had a major cost on the P. farnesiana groups suggest that the origin of the subgenus Acacia in the New World was in the drylands. subg. Of the seven morphological groups of Pseudomyrmex.984 sp/My. 2008..911 sp/My. Ingeae and Mimoseae belong to the subfamily Mimosoideae (Lewis et al. Conversely. acuifera clade (M in Figs.502 sp/My. with estimated ages of 10.. ferrugineus group which has a lower diversification rate and whose species have to developed physiological modifications (Kautz et al.068 sp/My in Coleoptera (Hunt et al.. Remarkably.97 and 12. Gómez-Acevedo et al. both groups originated in the Miocene (clades H.4–61. 2007) to 4. Also consistent with the increase in arid environments is the divergence age of the A.. Luckow et al. 2000). Ehrharta (Poaceae) with 0. constricta (Clarke et al.2 My. 2009). respectively (Table 5). macracantha and myrmecophilous species). Luckow et al. Acacia. 1979). Miller and Bayer. a Hawaiian cricket genus which has the highest rate recorded for the arthropods. In this study.98 My (clade C. farnesiana (Clarke et al. roigii). farnesiana. and the presence of extrafloral nectaries in their leaves. where differences in sexual behavior has been suggested as the forces that driven speciation (Mendelson and Shaw. the overall diversification rates obtained for the Neotropical genus Pseudomyrmex is 0.. Clarke et al. 2006). and phylogenetic analyses have shown that the tribe Ingeae is paraphyletic with respect to the Acacieae (Brown et al. 1 and 2 and Table 3). all inhabitants of arid regions in the Caribbean region (Clarke and Seigler. 2000.. similar habitats to those are preferred by the subgenus Acacia. Figs.06 ± 2. mainly with a Pacific islands distribution (Lewis et al. The subgenus Aculeiferum is pantropical. 2001.. Miller and Bayer. 100 species and two Neotropical genera: Myrcidris (2 species with scattered distributions in Brazil and Guyana) and Pseudomyrmex with ca. The monophyly of Acaciella is congruent with earlier studies (Clarke et al. 3 and 4). The section Filicinae. and we should argue than character 87 based on Ward and Downie (2005) should also need to be examined as a possible synapomorphy. with two basal and well-supported lineages (clades D. A. 3 and 4). (2009). Luckow et al. P. tenuissimus and P. In contrast. macracantha group was resolved as monophyletic. seasonally dry forests. macracantha group. P. picachensis with an estimated age in the Miocene (clade W. in our study the 39 sampled species of c. It is worth mention that Ward (1993) pointed out that males of the P. Brown et al. visco was established as not related to the rest of the species of this subgenus (clades O. The oldest lineage includes A. Gómez-Acevedo et al. the P. This latter hypothesis is congruent with the present study. the P. 2000). gaumeri. tenuis group and clade F representing a combination of ant generalists and all ant–plant mutualists. the estimated age of the node representing the separation of the Paleotropical (Tetraponera) from the Neotropical genera (Myrcidris + Pseudomyrmex) is of 95. in both the A. 2003. 2005).. Regarding to the nine morphological groups of Pseudomyrmex proposed by Ward (1989). str. ferrugineus. 1 and 2 and Table 3). ferrugineus group and P. In an earlier study (Ward and Brady.. Figs. lack of prickles and different eophyll sequence during seedling development within the rest of the subgenus Aculeiferum. 2000. 2006). reniformis. although the majority of them derived from a node situated at 8. 2003. in the New World could correspond with the Middle Miocene drylands of Mexico and Central America (clades O. lineage positioned in different sub-clades. Robinson and Harris. 2003). 90–100 My) and particularly by Pitman et al. Vassal. haytianus present similar diagnostic combination of character states. Morphological synapomorphies of Acaciella are lack of prickles and spines. P. albeit based on a smaller number of species: clade D comprising a still unassigned species. Filicinae. On previous morphological analysis. 2006. oculatus. remarkably does not grouped with the other species of the genus Acaciella. closely related to tribe Ingeae (Ariati et al. P. Robinson and Harris. (2006) have considered that species of the A. which recently was reinstated as Acaciella Britton and Rose (1928) by Rico-Arce and Bachman (2006). P. 2006). Moreover Seigler et al.. Figs. 2003. 1 and 2 and Table 3). 2003. These species are inhabitants of dry and seasonally dry forests. str. This genus has been supported by molecular and morphological analyses. (2003) proposed three evolutionary lineages within subgenus Aculeiferum: Acacia subg. gracilis. Figs. 2005). Luckow et al. lack of extrafloral nectaries. sericeus. The habitat preference of all species in the A. corresponding to the lower Cretaceous. 2000. Miller et al. 1 and 2 and Table 3). 2003. Other . 84–106 My). rufonigra) an age of 54 My was estimated for this divergence. Figs.. haytianus (clade L. Figs. Our study did not include morphological characters which could account for the differences between these two lineages. (1993. However in the present study A. 1999) was not so on a more recent molecular analysis (Ward and Downie. tenuis) were supported by phylogenetic analyses based on morphological and molecular data (Ward and Downie. found this group to be non-monophyletic.. placing Tetraponera species sister group to a clade grouping Myrcidris and Pseudomyrmex species (Figs. anomala is sister to these six species (clade P.. Based on morphological characters. 1 and 2 and Table 3). In contrast to Ward and Downie’s (2005) study. However. 2000. (2000). These species are characterized by having spicate inflorescences. coulteri group (13 species) are sufficiently distinct to be formalized into a new genus. Aculeiferum s. 3 and 4). 2003) and three in our study. and is consistent with the ages proposed by Goldblatt (1993. 4. Miller et al.80 ± 10.. P. Our results resolved that different species of A. considering only one species of Pseudomyrmex (P. only the P. Figs. / Molecular Phylogenetics and Evolution 56 (2010) 393–408 405 With respect to the A. The other six species of Acaciella were found to be monophyletic.. In addition. 2005). and possibly should be taken into consideration in the future delimitation of this group. (2000). In contrast. This age is similar to the divergence age ($100 My) given by Brady (2003) for African and South American soldier ants. 200 species (Ward and Downie. molecular evidence have placed Tetraponera as a sister group of Myrcidris + Pseudomyrmex (Ward and Downie.. 2003. 1045 species is largely confined to Australia. The present study is congruent with these findings. Figs. Figs. 200 species of Pseudomyrmex established as a monophyletic group (clade C. 2003. C. chamelensis. with a divergence age in the Pliocene (clade Q. pallidus. T. where A. a species not previously included in any previous molecular analyses. 95 My) for the separation of South America from Africa. The resulting divergence age placed the origin of this group in the Pliocene (clade K. Figs. 3 and 4). In the current study. and A. T. Predominantly Acaciella species inhabit Quercus-Pinus forests and to a lesser extent seasonally dry forests. McLoughlin (2001. P. and the Acacia coulteri group. Mariosousa Seigler and Ebinger. Miller and Bayer. seven of them (P. 1 and 2 and Table 3). viduus groups are found to be non-monophyletic. Robinson and Harris. Seigler et al. with fewer than 20 species outside this continent. A. with most of the recognized species groups by Ward (1989. Figs. 1 and 2 and Table 3). This estimated age does not support a role of a Gondwana vicariance. This group has been found to be non-monophyletic (Clarke et al. This species is nested within the clade representing the tribe Ingeae including the subgenus Phyllodineae (clade R. Previous molecular studies showed that Phyllodineae is a monophyletic subgenus. A. Murphy et al. 1972).82 Ma (clade A0 . subg. Character 121 from Ward and Downie’s study (2005) was argued by these authors to be shared only by species of this basal group of Pseudomyrmex (clade D). only two species were analyzed by Miller and Bayer (2001. 1 and 2 and Table 3). Ward and Downie (2005) have emphasized the importance of establishing the divergence age between the Paleotropical and Neotropical lineages of Pseudomyrmecinae.. 1991). Miller and Bayer. macracantha group is mesic forests. viduus group that was monophyletic on a previous morphological analysis (Ward. and dry scrub. Myrcidris established sister to the clade formed by Pseudomyrmex + Tetraponera (Ward. who included four species of this group. Contrastingly in our analysis. the group was considered as monophyletic by Jawad et al. pallens and the P.4. Aculeiferum sect. 2000). Systematics and phylogeny of Pseudomyrmex The subfamily Pseudomyrmecinae includes the Paleotropical genus Tetraponera with c. subg. Within clade F. ferrugineus group was found to be monophyletic. Aculeiferum s. and sister to one of the still unassigned species. 2005. This relationship was also found by Kautz et al. 4). Fig. 1 and 2 and Table 3). 2005).S. Figs. Clarke et al. In clade F. 2000. filiformis sister to species of the P. Luckow et al. 1 and 2 and Table 3).38 My (clade T. Our study included 12 species that grouped together and show a close relationship with species of the tribe Ingeae as well as with Acaciella chamelensis (clade S. and 8-grained polyads with a particular characteristic tectum (Rico-Arce and Bachman. oak forests. the P. This suggests that diversification of Aculeiferum s. houstoniana var. 2006. including 207 species. 1 and 2 and Table 3). comprises 15 Neotropical species. Figs.. 2000. berlandieri. pallens group was unresolved (Ward and Downie. four of the 13 species were included and these were resolved as paraphyletic (clade U. str. The subgenus Phyllodineae with c. gracilis) and one species of Tetraponera (T. F). 2005). American Journal of Botany 78. I. Lewis for comments to an early draft of this paper. 2005. Royal Botanic Gardens. P. Wanek. Acacia constricta (Fabaceae: Mimosoideae) and related species from the southwestern U. 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