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Phylogenetic reconstruction of Syntermitinae (Isoptera, Termitidae) based on morphological and molecular data

Phylogenetic reconstruction of Syntermitinae (Isoptera, Termitidae) based on morphological and... The subfamily Syntermitinae comprises a group of Neotropical termites with 18 genera and 101 species described. It has been considered a natural group, but relationships among OPENACCESS the genera within the subfamily remain uncertain, and some genera appear to be non-mono- Citation: Rocha MM, Morales-Corrêa e Castro AC, phyletic. Here, we provide a comprehensive phylogeny including six Neotropical species Cuezzo C, Cancello EM (2017) Phylogenetic reconstruction of Syntermitinae (Isoptera, of Termitinae as outgroup, 42 Syntermitinae species as ingroup, 92 morphological charac- Termitidae) based on morphological and ters (from external and internal anatomy of soldier and worker castes) and 117 molecular molecular data. PLoS ONE 12(3): e0174366. sequences (109 obtained for this study and 8 from GenBank) of 4 gene regions (41 and 22 https://doi.org/10.1371/journal.pone.0174366 from Cytochrome Oxidase I and II respectively, 19 from Cytochrome b, and 35 from 16S Editor: Tony Robillard, Museum National d'Histoire rDNA). Morphological and molecular data were analyzed in combination, with the Bayesian Naturelle, FRANCE inference method, and the important aspects of termite biology, defense and feeding habits Received: May 18, 2016 are discussed based on the resulting tree. Although useful for providing diagnostic charac- Accepted: March 8, 2017 ters, the morphology of the soldier caste reveals several cases of convergence; whereas Published: March 22, 2017 the feeding habit shows indications of evolutionary significance. Copyright:© 2017 Rocha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Introduction reproduction in any medium, provided the original author and source are credited. The subfamily Syntermitinae comprises a group of Neotropical termites that ranges from southern Mexico (Cahuallitermes) to northern Argentina (Cornitermes, Procornitermes, Data Availability Statement: All relevant data are within the paper and its Supporting Information Rhynchotermes, Syntermes), with the richest generic and specific diversity in the Brazilian Cer- files. rado biome. Fifteen syntermitine genera occur in the Cerrado, where several species of Corni- termes, Silvestritermes and Syntermes construct conspicuous epigeal nests that characterize this Funding: We received support from the São Paulo Research Foundation, Brazil (FAPESP) [http://www. savanna-like landscape. Cornitermes cumulans can reach a nest density of 55/ha, and is consid- fapesp.br/], grants 2012/00952-9 to MMR, 2013/ ered a keystone species in the Cerrado [1]. These termite nests may harbor many other termite 20068-9 to EMC and 2013/05610-1 to CC, and species as well as other groups of invertebrates. from the National Council for Scientific and The feeding and nesting habits of syntermitine species are diverse. The group includes Technological Development, Brazil (CNPq) [http:// grass/litter-feeders, intermediate feeders, and humus-feeders. The nests are variable; some spe- cnpq.br/], grant 308227/2013-0 to EMC. The funders had no role in study design, data collection cies build earthen nests; most are commonly epigeal, but arboreal and subterranean forms are PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 1 / 29 Phylogenetic reconstruction of Syntermitinae and analysis, decision to publish, or preparation of well known. Other nesting habits include inquilines, reformers, and diffuse galleries in the the manuscript. ground. A total of 18 genera and 101 species are now established as part of the subfamily. Some of Competing interests: The authors have declared that no competing interests exist. the taxa treated in taxonomic revisions and original descriptions in the last 20 years are Acan- gaobitermes [2], Armitermes [3], Cahuallitermes [4], Cyrilliotermes [5], Curvitermes [6], Labio- termes [7], Macuxitermes [8, 9], Noirotitermes [10], Paracurvitermes [11], Rhynchotermes [12], and Syntermes [13]. However, the status of Embiratermes is still in need of revision [3]. Engel and Krishna [14] proposed the subfamily, including only Cornitermes, Labiotermes, Procornitermes and Syntermes. Lately, Constantino and Carvalho [11] gave a new diagnosis, considering all the genera of ªmandibulate nasutesº then described (those genera with soldiers having developed mandibles and a recognizable frontal tube). Although Syntermitinae is a recently proposed taxonomic category, the ªmandibulate nasutesº group was recognized very early in the termite literature. The ªmandibulate nasutesº together with ªtrue nasutesº (group of genera with soldiers having vestigial mandibles and a developed frontal tube) were consid- ered part of the worldwide subfamily Nasutitermitinae. In the last century, two hypotheses were proposed regarding the origin of the nasute soldier: the monophyletic hypothesis, where the ªmandibulate soldiersº form an ancestral group of the ªtrue nasutesº [15, 16]; and the diphyletic hypothesis, where two independent lineages of ªmandibulate soldiersº led to the ªtrue nasutesº [17±20]. Inward and collaborators [21], in a comprehensive phylogenetic analysis with morphologi- cal and molecular data, supported the hypothesis that ªmandibulate nasutesº and ªtrue nasutesº are two distinct, independent lineages, and that the Syntermitinae is more closely related to the Amitermes-group (Termitinae) than to the Nasutitermitinae. Rocha and collaborators [3] developed a revisionary proposal for the genus Armitermes “sensu lato”, which included a cladistic analysis involving morphological characters from all species of Armitermes and representatives of all genera of ªmandibulate nasutesº. In this phylo- genetic approach, the genus Armitermes appears as polyphyletic, and some species are relo- cated to new genera, although the relationships among Syntermitinae genera are poorly resolved. Herein, we propose a comprehensive phylogenetic hypothesis for Syntermitinae, based on combined morphological and molecular data under a Bayesian approach; and reconstruct some aspects of the defense behavior and feeding habits of the group. Material and methods Taxon sampling and outgroup selection We included a total of 42 syntermitine species as ingroup, representing the diversity of the 18 currently described syntermitine genera; and 6 species of Termitinae as outgroup, chosen for their established relationships to Syntermitinae [21±23] and also based on our experience with Neotropical termites. Morphological studies were carried out on termite specimens deposited in the Isoptera collection of the Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil (MZUSP). A representative sample of each lot used to perform the molecular studies was formally deposited in the MZUSP as well and appropriately registered for public consult. Morphological characters We included a total of 92 characters, 40 of the soldier external morphology, 42 of the coiling gut in situ and the configuration of the different parts of the worker digestive tube, and 10 of worker external morphology. The morphological character data are expanded from our previ- ous study [3]; most characters are referenced in Figs 1±17. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 2 / 29 Phylogenetic reconstruction of Syntermitinae Fig 1. Examples of shapes of labrum. A. Syntermes molestus; B. Procornitermes triacifer; C. Microcerotermes strunckii; D. Labiotermes labralis; E. Cornitermes cumulans F. Silvestritermes holmgreni. https://doi.org/10.1371/journal.pone.0174366.g001 Fig 2. Examples of shapes of postmentum. A. Cornitermes cumulans; B. Labiotermes labralis; C. Rhynchotermes nasutissimus. https://doi.org/10.1371/journal.pone.0174366.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 3 / 29 Phylogenetic reconstruction of Syntermitinae Fig 3. Examples of types of frontal gland openings and frontal tube shapes. A. Microcerotermes strunckii; B. Amitermes amifer; C. Syntermes molestus; D. Labiotermes labralis; E. Procornitermes araujoi; F. Embiratermes festivellus. https://doi.org/10.1371/journal.pone.0174366.g003 The character matrix (S1 Table) was edited and managed with Mesquite v.3.04 [24]. Soldier head. 01. Labrum, hyaline tip: (0) absent (Fig 1C and 1F); (1) present (Fig 1A, 1B, 1D and 1E). PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 4 / 29 Phylogenetic reconstruction of Syntermitinae Fig 4. Examples of shapes of outer margins of the forecoxae and projections. A. Syntermes molestus; B. Cornitermes cumulans; C. Armitermes spininotus; D. Embiratermes festivellus; E. Rhynchotermes nasutissimus. https://doi.org/10.1371/journal.pone.0174366.g004 02. Shape of hyaline tip: (0) flat (Fig 1A and 1D); (1) fingerlike (Fig 1B and 1E). 03. Silhouette in dorsal view: (0) cuspidate (Fig 1A, 1B and 1E); (1) lanceolate (Fig 1C); (2) obtuse (Fig 1D and 1F). 04. Cuspidate margins: (0) slender (Fig 1A and 1E); (1) clearly angulate (Fig 1B). 05. Postmentum lateral margins: (0) angled (Fig 2A); (1) sinusoidal (Fig 2B); (2) convex (Fig 2C). 06. Postmentum length: (0) elongated (Fig 2A and 2B); (1) shorter (Fig 2C). 07. Shape of head in dorsal view: (0) rectangular, elongated; (1) rectangular, sort; (2) rounded. Fig 5. Examples of lateral lobes of the pronotum. A. Labiotermes labralis; B. Embiratermes festivellus; C. Syntermes molestus. https://doi.org/10.1371/journal.pone.0174366.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 5 / 29 Phylogenetic reconstruction of Syntermitinae Fig 6. Examples of shapes of thoracic nota. A. Labiotermes labralis; B. Armitermes spininotus; C. Syntermes molestus; D. Syntermes crassilabrum. https://doi.org/10.1371/journal.pone.0174366.g006 08. Number of antennal articles: (0) 20; (1) 19; (2) 18; (3) 17; (4) 16; (5) 15; (6) 14; (7); 13; (8); 12 (9) 11. 09. Head capsule microsculpture: (0) absent; (1) present (character 7 of [3]). 10. Visibility of frontal pore aperture: (0) indistinct (Fig 3A); (1) distinct (Fig 3B±3F). 11. Frontal pore shape: (0) retracted and narrow (Fig 3B); (1) protruded and wide (Fig 3C± 3F). 12. Membranous tissue at frontal pore aperture: (0) absent (Fig 3B and 3C); (1) present (Fig 3D±3F). Fig 7. Examples of mandibles (right) and molar regions. A. Procornitermes araujoi; B. Embiratermes festivellus; C. Curvitermes odontognathus. (Arrows indicate the molar region). https://doi.org/10.1371/journal.pone.0174366.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 6 / 29 Phylogenetic reconstruction of Syntermitinae Fig 8. Examples of pulvilli ornamentations. A. Uncitermes teevani; B. Mapinguaritermes peruanus. https://doi.org/10.1371/journal.pone.0174366.g008 13. Frontal projection: (0) absent (Fig 3A±3C); (1) present (Fig 3D and 3E). 14. Length of frontal tube: (0) reaching clypeus (Fig 3E); (1) surpassing clypeus (Fig 3F); (2) lump in profile (Fig 3D); (3) salience (Fig 3C). Soldier thorax. 15. Row of stout bristles on outer margins of forecoxae: (0) absent; (1) present (character 29 of [3]). 16. Stout bristles along femur: (0) absent; (1) present (as described for Labiotermes [7]). 17. Ornaments on internal face of tibia: (0) flat; (1) row of 20 spines. 18. Tibial spurs formula: (0) 3:2:2; (1) 2:2:2 19. Shape of outer margin of forecoxae: (0) nearly straight (Fig 4A); (1) with lump (Fig 4B). 20. Outer margin of forecoxae, distal portion: (0) round (Fig 4A and 4B); (1) keeled (Fig 4C); (2) spiniform (Fig 4D). 21. Spine on proximal portion of coxae: (0) absent (Fig 4A±4D); (1) present (Fig 4E). 22. Lateral lobes of pronotum: (0) not projected (Fig 5A); (1) slightly projected (Fig 5B); (2) well projected (Fig 5C). Fig 9. Insertion of the stomodeal valve in the mesenteron (A, B) and examples of alignment of the mesenteric tongues (C±F). A. Cornitermes cumulans; B. Procornitermes striatus; C. Silvestritermes holmgreni; D. Mapinguaritermes peruanus; E. Rhynchotermes nasutissimus; F. Ibitermes curupira. https://doi.org/10.1371/journal.pone.0174366.g009 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 7 / 29 Phylogenetic reconstruction of Syntermitinae Fig 10. Homology between the ornament regions inside the first proctodeal segment (red, central area; yellow, distal area; and green, region around the mesenteric tongue). A Curvitermes odontognathus; B. Embiratermes festivellus; C. Cornitermes cumulans. https://doi.org/10.1371/journal.pone.0174366.g010 23. Lateral margins of lobes (pronotum): (0) rounded (Fig 6A); (1) angulate (Fig 6C); (2) acuminate (Fig 6B and 6D). 24. Lateral margins of lobes (mesonotum): (0) rounded (Fig 6A); (1) angulate (Fig 6B and 6C); (2) acuminate (Fig 6D). 25. Lateral margins of lobes (metanotum): (0) rounded (Fig 6A and 6B); (1) angulate (Fig 6C); (2) acuminate (Fig 6D). 26. Outer margin of pronotum: (0) smooth (Fig 6A, 6C and 6D); (1) serrated (Fig 6B). 27. Outline of margins of metanotum and mesonotum: (0) smooth (Fig 6C and 6D); (1) ser- rated (Fig 6A and 6B). Soldier mandibles. 28. Molar region: (0) indistinct (Fig 7A); (1) distinct (Fig 7B and 7C). Fig 11. Examples of transition from tubular portion to dilated portion in P1 (arrows, initial portion of the dilated regions). A. Silvestritermes holmgreni; B. Cornitermes cumulans; C. Cyrilliotermes angulariceps; D. Uncitermes teevani. https://doi.org/10.1371/journal.pone.0174366.g011 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 8 / 29 Phylogenetic reconstruction of Syntermitinae Fig 12. Examples of connections between P1 and P3 through P2. A. Silvestritermes holmgreni; B. Cyrilliotermes angulariceps; C. Embiratermes festivellus. https://doi.org/10.1371/journal.pone.0174366.g012 29. When distinct, relative size of molar region: (0) reduced (Fig 7B); (1) developed (Fig 7C). 30. First marginal tooth of left mandible: (0) absent; (1) present 31. Second marginal tooth of left mandible: (0) absent; (1) present. 32. Shape between first and second marginal teeth of left mandible: (0) ªVº concavity; (1) cutting edge Fig 13. Examples of P2 insertion, relative to abdomen length (arrows, P2 position). A. Silvestritermes holmgreni; B. Cyrilliotermes angulariceps. https://doi.org/10.1371/journal.pone.0174366.g013 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 9 / 29 Phylogenetic reconstruction of Syntermitinae Fig 14. Examples of enteric valve shapes. A. Amitermes amifer; B. Curvitermes odontognathus; C. Mapinguaritermes peruanus; D. Genuotermes spinifer; E. Embiratermes festivellus; F. Embiratermes silvestrii; G. Procornitermes lespessi; H. Cornitermes cumulans; I. Silvestritermes holmgreni. https://doi.org/10.1371/journal.pone.0174366.g014 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 10 / 29 Phylogenetic reconstruction of Syntermitinae Fig 15. Examples of P3b shapes and isthmus insertions (arrow, sub-apical P3 insertion). A. Microcerotermes strunckii; B. Curvitermes odontognathus; C. Embiratermes ignotus; D. Acangaobitermes krishnai. https://doi.org/10.1371/journal.pone.0174366.g015 33. Shape of cutting edge between first and second marginal teeth of left mandible: (0) smooth; (1) serrated. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 11 / 29 Phylogenetic reconstruction of Syntermitinae Fig 16. Examples of body proportions and profiles. A. Cornitermes cumulans; B. Labiotermes labralis; C. Acangaobitermes krishnai. https://doi.org/10.1371/journal.pone.0174366.g016 34. First marginal tooth of right mandible: (0) absent; (1) present 35. Second marginal tooth of right mandible: (0) absent; (1) present. 36. Position of second marginal tooth: (0) in proximal portion; (1) in middle. 37. Shape of apical tooth: (0) slender; (1) strong; (2) sinuous. 38. Apical tooth curvature: (0) strongly arched; (1) slightly arched. 39. Internal outline of apical tooth: (0) concave (Fig 7B); (1) sinusoidal (Fig 7A). 40. Subapical tooth: (0) present; (1) absent. Characters of the gut anatomy. 41. Gizzard: Ornamentation of first-order pulvilli: (0) without notable ornaments (Fig 8A); (1) with developed spines (Fig 8B). 42. Insertion of stomodeal valve in mesenteron: (0) apical (Fig 9A); (1) subapical (Fig 9B). 43. Mesenteric tongue: (0) absent; (1) present. 44. Mesenteric tongue, proximal portion: (0) robust (Fig 9C, 9D and 9F); (1) constricted (Figs 9E and 11B); (2) filiform. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 12 / 29 Phylogenetic reconstruction of Syntermitinae Fig 17. Worker mandibles (not to same scale). A. Microcerotermes strunckii; B. Cornitermes cumulans; C. Silvestritermes holmgreni; D. Curvitermes odontognathus; E. Paracurvitermes manni; F. detail of C. cumulans molar plate notch; G. detail of S. holmgreni molar plate notch. https://doi.org/10.1371/journal.pone.0174366.g017 45. Alignment of mesenteric tongue: (0) continuous with external face of the mesenteric arch (Fig 9C); (1) with apex turned (Fig 9D); (2) laterally to mesenteric arch (Fig 9); (3) twisted (Fig 9F). 46. Secondary mesenteric tongue: (0) absent; (1) present. 47. Malpighian tubules attachment: (0) in two pairs; (1) four united. 48. Large ampulla at insertions of Malpighian tubules: (0) absent; (1) present. 49. Position of Malpighian tubules: (0) internal to mesenteric arch; (1) external to mesen- teric arch. Internal ornamentation of first proctodeal segment. The P1 internal ornaments were described by Rocha and Constantini [25], and the homology between the regions adopted in this study is explained in Fig 10 [central area in red, distal area in yellow, and around the mesenteric tongue(s) in green]. 50. Ornaments: (0) absent; (1) present. 51. Type of spines covering central area (0) Small spines in rows; (1) aciculiform; (2) robust spines; (3) as thin setae; (4) trifurcated spines. 52. Degree of sclerotization of spines: (0) slightly sclerotized; (1) strongly sclerotized. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 13 / 29 Phylogenetic reconstruction of Syntermitinae 53. Pattern of spine coverage in central area: (0) transverse; (1) longitudinal; (2) spaced. 54. Position of spines of central area, relative to mesenteric tongue: (0) after mesenteric ton- gue; (1) lateral to mesenteric tongue. 55. Central ridges: (0) absent; (1) present. 56. Spines around mesenteric tongue: (0) absent; (1) present. 57. Type of spines around mesenteric tongue: (0) single; (1) in small rows. 58. Coverage in distal area: (0) absent; (1) present. 59. Pattern of coverage in distal area: (0) sparse; (1) grouped in three areas. 60. When grouped in three areas: (0) complete columns; (1) incomplete columns; (2) rounded areas. First proctodeal segment. 61. Shape of P1: (0) tubular; (1) dilated. 62. Length of P1 relative to abdomen: (0) nearly the same (relatively outstretched in the abdomen); (1) longer (relatively coiled inside the abdomen). 63. Shape of dilated portion: (0) fusiform; (1) globose. 64. Transition from tubular portion to dilated portion of P1, see arrows: (0) distally con- stricted (Fig 11A); (1) gradual (Fig 11B); (2) proximally constricted (Fig 11C); (3) very strangu- lated (Fig 11D). 65. P1 orientation in relation to body axis: (0) parallel (Fig 11A); (1) diagonal (Fig 11B). 66. Shape of P1 final portion: (0) tubular (Fig 12A); (1) conical (Fig 12C); (2) tubular and narrow. 67. Shape of tubular P1 final portion: (0) arched; (1) straight. Second proctodeal segment. 68. Position of P2 insertion relative to abdomen length: (0) distally (Fig 13A); (1) at midlength (Fig 13B). 69. Position of P2 insertion in dorsal view: (0) on left side of body; (1) on right side of body. 70. Symmetry of enteric valve armature: (0) hexa-radial; (1) tri-radial; (2) asymmetric. 71. Category of ridges: (0) large pads (Fig 14A); (1) simple ridge (Fig 14B); (2) ridge slightly dilated at apex (Fig 14C); (3) finger-like (Fig 14D±14F); (4) lobate (Fig 14G and 14H); (5) bul- bous (Fig 14I). 72. Type of finger-like ridge: (0) protruded (Fig 14D); (1) oblong (Fig 14E); (2) conical (Fig 14F). 73. Type of lobate ridge: (0) slightly lobed (Fig 14G); (1) auricular (Fig 14H). 74. Proportions of lobate ridge: (0) equal; (1) unequal. 75. When slightly dilated at apex, length: (0) elongated; (1) short. 76. Ornaments: (0) absent; (1) present. 77. Ornament coverage: (0) triangular; (1) aciculiform. Third proctodeal segment. 78. Initial portion of P3: (0) directly connected (Fig 12); (1) bottleneck (Fig 12B); (2) well-developed enteric valve seating (Fig 12C). 79. Smooth diverticulum of P3a: (0) absent; (1) present. 80. P3b shape in dorsal view: (0) globose (Fig 15B); (1) arched (Fig 15C and 15D); (2) not protruded (Fig 15A). 81. Direction of P3b when arched: (0) turned forward (Fig 15D); (1) turned to right side of body (Fig 15C). 82. Insertion of isthmus: (0) apical (Fig 15A and 15B); (1) sub-apical (Fig 15C and 15D, arrow). Characters based on external morphology of workers. 83. Size proportion of head to thorax: (0) head much larger than thorax (Fig 16A); (1) head size similar to thorax (Fig 16B and 16C). 84. Body in profile: (0) slender (Fig 16C); (1) waisted (Fig 16A and 16B). PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 14 / 29 Phylogenetic reconstruction of Syntermitinae 85. Mandibles: Relative size of left apical tooth: (0) smaller than M1 (Fig 17B); (1) equal to M1 (Fig 17A and 17C); (2) more prominent than M1 (Fig 17D and 17E). 86. Edge of apical tooth: (0) straight (Fig 17A, 17B and 17C); (1) concave (Fig 17D and 17E). 87. M3 tooth on left mandible: (0) present, conspicuous (Fig 17A); (1) present, reduced (Fig 17B±17E). 88. M2 tooth on right mandible: (0) present, conspicuous (Fig 17A±17C); (1) present, reduced (Fig 17D); (2) absent (Fig 17E). 89. Relative position of right M2 tooth: (0) near middle of M1 and molar plate (Fig 17C); (1) near M1 tooth (Fig 17A and 17D); (2) near molar plate (Fig 17B); (3) fused to M1 (Fig 17E). 90. Right M2 posterior margin: (0) straight; (1) concave. 91. Molar plate notch: (0) absent; (1) present, 90 degrees (Fig 17F, arrow); (2) present, more than 90 degrees (Fig 17G, arrow). 92. Molar region: (0) with ridges; (1) with reduced ridges; (2) without ridges. Molecular protocols We chose four regions of the mitochondrial genome, Cytochrome Oxidase I and II (COI ~600 bp, COII ~660 bp), Cytochrome b (Cyt B ~340 bp) and 16S rDNA (~430 bp). The DNA was extracted preferentially from the head and thorax of a single soldier individual preserved in 95% ethanol (Table 1), with the set of reagents from the DNeasy Blood & Tissue Kits (Qiagen), supplemented with 20 mg/ml proteinase K, following the manufacturer's protocol. The homogenates were incubated at 55ÊC for 3 h. The gene fragments were then amplified by poly- merase chain reaction, PCR [26]. The primers and the amplification conditions are listed in Table 2. PCR was performed in 25 μL reactions (12.5 μL PCR master mix Promega1, 0.6 μM of each primer, 3.0 μL of total DNA, and 3.5 μL deionized water). The amplified PCR products were determined by gel electrophoresis on a 1% agarose gel diluted in TAE buffer (1X) (0.04 M Tris base, 0.02 M acetic acid, and 1 mM EDTA). This same buffer was also used in 1-h elec- trophoresis runs in an 8-V/cm length gel. All reaction products were purified with Wizard1 SV Gel and PCR Clean-Up System (Promega), following the manufacturer's protocols. Puri- fied PCR products were sequenced with the same primers used in the original PCR reactions and the BigDye1 Terminator v3.1 Cycle Sequencing Kit, under the same conditions of PCR. The sense and antisense sequences obtained from each amplicon were assembled, and a con- sensus sequence for each gene was generated with Geneious v.8.1.7 analysis tools [27]. The nucleotide sequences reported here were submitted to the GenBank database under the acces- sion numbers indicated in Table 1. Analyses The saturation of the molecular data was assessed with DAMBE v.6.0.48 [33] using the test of substitution saturation by [34, 35]. The saturation test showed little saturation, indicating that the data were suitable for phylogenetic analysis (Iss < ISSc; p < 0.05). To evaluate the most useful data set, we made several tests combining different sets of sequences (the four gene sequences, COII + 16S rDNA + COI, COII + 16S rDNA + Cytb and only COII + 16S rDNA), with and without the morphological data, and with the protein-cod- ing sequences partitioned either by genes or by the codon position. The results are summa- rized in the Table 3. The models for each DNA data partition were determined using the JModel Test 2 [36] and PartitionFinder v1.1.0 [37], for the morphological data partition the states were unordered. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 15 / 29 Phylogenetic reconstruction of Syntermitinae Table 1. List of GenBank accession codes for each gene. MZUSPLot no. COII COI Cytb 16S rDNA Termitinae Amitermes amifer 23727 KX247014 n.d. n.d. KX247076 Amitermes nordestinus 16373 KX247015 n.d. n.d. KX247077 Cylindrotermes parvignathus 23881 *DQ442113.1 n.d. KX247054 KX247075 Genuotermes spinifer 16354 KX247016 KY379279 n.d. KX247078 Microcerotermes sp. 21513 KX247013 n.d. n.d. KX247074 Orthognathotermes sp. 16233 * DQ442213 n.d. n.d. KX247073 Syntermitinae Acangaobitermes krishnai 13670 n.d. n.d. n.d. KX247081 Armitermes spininotus 24420 KX247034 n.d. KX247062 KX247092 Cahuallitermes intermedius 15463 KX247037 n.d. n.d. n.d. Cornitermes acignathus 24421 KX247039 KX247051 n.d. n.d. Cornitermes bequaerti 15970 KY379285 KY379284 KX247065 KX247097 Cornitermes bolivianus 20596 n.d. KX247052 KX247067 KY379286 Cornitermes cumulans 24423 ** EU253899 ** EU253860.1 KX247064 KX247096 Cornitermes ovatus 20617 KX247038 n.d. KX247066 KX247098 Cornitermes silvestrii 16232 KX247040 KY379283 n.d. KX247099 Curvitermes odontognathus 20705 KX247018 n.d. n.d. KX247080 Cyrilliotermes angulariceps 20709 KX247027 KY379282 KX247061 n.d. Embiratermes brevinasus 24424 KX247022 n.d. KX247057 KX247083 Embiratermes festivellus 24425 KX247026 KY379281 KX247060 KX247085 Embiratermes heteropterus 24427 KX247035 n.d. n.d. KX247093 Embiratermes ignotus 20810 KX247020 KX247048 KX247055 n.d. Embiratermes neotenicus 23830 KX247025 KX247050 KX247059 n.d. Embiratermes silvestrii 24428 KX247021 KY379280 KX247056 KX247082 Ibitermes curupira 24429 KX247029 n.d. n.d. KX247087 Labiotermes emersoni 16219 KX247030 KY379278 n.d. KX247088 Labiotermes labralis 14771 KX247031 n.d. n.d. KX247089 Labiotermes leptothrix 20997 KX247032 n.d. n.d. KX247090 Labiotermes orthocephalus 14829 KX247033 n.d. n.d. KX247091 Macuxitermes triceratops 16103 n.d. n.d. n.d. KX247095 Mapinguaritermes peruanus 14490 KX247028 n.d. n.d. KX247086 Noirotitermes noiroti 24430 KX247019 n.d. n.d. n.d. Paracurvitermes manni 21026 KX247017 KY379277 n.d. KX247079 Procornitermes araujoi 16315 ** EU253902 ** EU253862 n.d. n.d. Procornitermes lespesii 24431 KX247041 KX247053 KX247068 n.d. Procornitermes triacifer 24432 n.d. KY379276 KX247069 KX247100 Rhynchotermes nasutissimus 15981 KX247042 n.d. n.d. KX247101 Rhynchotermes perarmatus 24433 KX247043 KY379275 n.d. KX247102 Silvestritermes holmgreni 20549 KX247023 KX247049 KX247058 n.d. Silvestritermes minutus 20553 KX247024 KY379274 n.d. KX247084 Syntermes crassilabrum 21044 KX247046 KY379273 KX247071 KX247105 Syntermes grandis 16338 ** EU253903 ** EU253863 n.d. n.d. Syntermes molestus 21069 KX247045 KY379272 KX247070 KX247104 Syntermes parallelus 14753 KX247047 n.d. KX247072 KX247106 Syntermes spinosus 21155 KX247044 n.d. n.d. KX247103 (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 16 / 29 Phylogenetic reconstruction of Syntermitinae Table 1. (Continued ) MZUSPLot no. COII COI Cytb 16S rDNA Uncitermes teevani 20574 KX247036 n.d. KX247063 KX247094 Sequences obtained from other papers are indicated by the asterisks * [21] ** [23], n.d.: no data. https://doi.org/10.1371/journal.pone.0174366.t001 The Bayesian inference analyses were performed with MrBayes version 3.2.1 [38], in the CIPRES Science Gateway V. 3.3 [39]; in all analyses, four chains were run for 50 million gener- ations and sampled every thousand generations (two runs). In all cases the burn-in limitation was determined by visual inspection of the trace-plot and evaluation of the effective sample size value (ESS) of the combined runs, using Tracer v1.6 [40]. The burn-in of 1% was sufficient. Ancestral character states were reconstructed with the help of Mesquite v.3.04 [24], by the parsimony criterion. Results and discussion From the total of 48 species used in our analyses, we obtained DNA data from three or four different gene for 25 species, two different sequences for 16 species and only one sequence for 4 species (Table 1). Two taxa are represented only by morphological data. About one third of the sequences information is absent. Although the poverty of sequences may compromise the results, the majority of taxa share COII and 16s rDNA information (The information for COII sequences is absent only in four taxa and for 16S rDNA in 10), the major part of lacking infor- mation is concentrated in COI and Cytb sequences. Among the trees obtained, three of them present informative topology (with few poly- tomies) and high posterior probabilities (especially in the basal nodes); 1)The result of an anal- ysis with Morphology + COII + 16S rDNA, partitioned by genes (COII: GTR +I + G, 16S rDNA: GTR +G) represented in Fig 18 and Fig 20A (29 nodes more than 0.9, 2 nodes equal Table 2. Sequences of primers and PCR profiles used. Gene Primer Sequence (5'! 3') Reference COI F-LCO GGTCAACAAATCATAAAGATATTGG [28] R-HCO TAAACTTCAGGGTGACCAAAAAATCA [28] COII F-Leu TCTAATATGGCAGATTAGTGC [29] R-Lys GAGACCAGTACTTGCTTTCAGTCATC [29] Cyt B cytb612 CCATCCAACATCTCCGCATGATGAAA [30] cytb920 CCCTCAGAATGATATTTGGCCTCA [30] 16S rDNA 16SAr CGCCTGTTTATCAAAAACAT [31] 16SF TTACGCTGTTATCCCTAA [32] Conditions Gene Heat Denaturation Annealing Extension Final extension Cycles COI 94ÊC (2 min) 94ÊC (1 min) 43ÊC (1 min)* 72ÊC (1 min 15 s) 72ÊC (7 min) 40 COII 94ÊC (2 min) 94ÊC (1 min) 45 to 53ÊC (1 min)* 72ÊC (1 min 15 s) 72ÊC (7 min) 40 Cyt B 94ÊC (2 min) 94ÊC (1 min) 50ÊC (1 min) 72ÊC (1 min 15 s) 72ÊC (7 min) 40 16S rDNA 94ÊC (2 min) 94ÊC (1 min) 50ÊC (1 min) 72ÊC (1 min 15 s) 72ÊC (7 min) 40 *every 2ÊC, the temperature was maintained for 30 s. https://doi.org/10.1371/journal.pone.0174366.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 17 / 29 Phylogenetic reconstruction of Syntermitinae Table 3. Summarized results of combining different sets of sequences and types of codification for protein-coding sequences and their respective Estimated sample sizes (ESS) of each run combined. COII; COI; Cytb; 16S rDNA COII; COI; 16S rDNA COII; Cytb; 16S rDNA COII;16S rDNA Without the morphological data Protein-coding S13 Fig S10 Fig S16 Fig S6 Fig sequences partitioned by genes ESS: 60648 ESS: 63697 ESS: 56201 ESS: 50544 Without the morphological data Protein-coding S12 Fig S9 Fig S15 Fig S5 Fig sequences partitioned by the codon position ESS: 58695 ESS: 60012 ESS:54663 ESS:51510 With morphological data Protein-coding S11 Fig S8 Fig S14 Fig Fig 18 sequences partitioned by genes ESS: 56726 ESS: 56635 ESS: 60688 ESS: 65654 With morphological data Protein-coding Fig 19 S7 Fig Fig 20 S4 Fig sequences partitioned by the codon position ESS: 56656 ESS: 55645 ESS: 56361 ESS: 57063 https://doi.org/10.1371/journal.pone.0174366.t003 0.89, and 7 nodes less than 0.8); 2) The result of Morphology + all sequences, with protein-cod- ing genes partitioned by codons (1st codon: GTR+G, 2nd codon: HKY+I+G, 3rd codon: GTR +I+G, 16S rDNA: GTR+I+G), represented in Fig 19 and Fig 20B (26 nodes more than 0.9, 4 nodes between 0.8 and 0.9, and 10 nodes less than 0.8); 3) The result of Morphology COII + 16S rDNA + Cytb, with protein-coding genes partitioned by codons (1st codon: SYM+I+G, 2nd codon: HKY+I+G, 3rd codon: GTR+I+G, 16S rDNA: GTR+I+G), represented in Fig 20 and Fig 20C (30 nodes more than 0.9, 3 nodes between 0.8 and 0.9, and 9 nodes less than 0.8). Their respective traceplots are illustrated in S1±S3 Figs. Considering all obtained trees (Figs 18±20 and S4±S16 Figs), the exclusion of morphological data from the analysis result in large pectinate nodes (S5, S6, S9, S10, S12, S13, S15 and S16 Figs). The position of Acangaobitermes krishnai, Cahuallitermes intermedius, Macuxitermes tricer- atops and Noirotitermes noiroti represented by only one sequence and Armitermes armiger and Ibitermes tellustris, with no sequences, remains stable in the three more consistent trees (Fig 21), and considering just morphological characters, mainly from internal morphology, the position among them seems reliable. Comparing the three most robust results (Fig 21A±21C), their topologies have a few diver- gences; the groups of genera (delimited by the best-supported basal nodes, and indicated by the same colors in each tree) were recovered with identical taxa compositions. The more notable differences among the selected trees are: The position of the genus Syn- termes (the red branch, with taxa initiated by the acronym ªSYº), not resolved in tree A and positioned in trees B and C as a sister group of the yellow branch, composed of Rhynchotermes (indicated by the acronym ªRHº), Procornitermes (PR), Cahuallitermes (CA) and Cornitermes (CO). The relationships among the taxa are indicated by dark-green branches; Genuotermes (GESP), Curvitermes (CUOD), Embiratermes (EM), Paracurvitermes (PAMA), Cyrilliotermes (CYAN) and Silvestritermes (SI); possible paraphyletic in trees A and B, and recovered as a monophyletic group in tree C. Although these results do not contradict each other, for prudence we opted to reconstruct and discuss the ancestral character states using tree A, which is less resolved but more conservative. Taxonomic discussion The sample of species is sufficiently comprehensive to allow a discussion that previous studies did not attempt. Four genera appeared as paraphyletic in our analysis: Armitermes (possibly), Procornitermes (possibly), Embiratermes, and Ibitermes. The cases of Armitermes (pink branch, Fig 21) and Procornitermes (part of the yellow branch, Fig 21) can be resolved with reallocation of a few species: for Armitermes the most con- servative solution is include all taxons (Armitermes, Macuxitermes, Uncitermes and PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 18 / 29 Phylogenetic reconstruction of Syntermitinae Fig 18. Tree obtained with the Bayesian analysis with morphological data and COII, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, and the branch color represents the posterior probability. https://doi.org/10.1371/journal.pone.0174366.g018 Embiratermes heterotypus) into a single genus, since the node that it is grouping the five taxa has a high posterior probability. Nevertheless, considering we only obtain one sequences for Macuxitermes, and also the support for the three more internal nodes are low, we think more studies are necessary to infer consistently the relationship among the taxa before introducing nomenclatural changes; for Procornitermes, resurrecting it from Triacitermes Emerson, now including Procornitermes triacifer and P. araujoi is a admissible solution, however the para- phyly of the genus is not a consensus among the results. More detailed studies for these cases are necessary before formal proposals for nomenclatural changes can be made. A revisionary work is necessary in the next future to reassess generic and specific limits as well as the intergeneric relationships of Embiratermes and Ibitermes within other members of the subfamily, since their named species used herein as terminals are spread all over the tree. A surprising result is the position of Genuotermes, deeply inserted in our tree. Although the association with Syntermitinae is unintuitive, some unique characteristics are shared with PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 19 / 29 Phylogenetic reconstruction of Syntermitinae Fig 19. Tree obtained with the Bayesian analysis with morphological data and all four sequences, partitioned by codons. The respective posterior probability is indicated above each node, and the branch color represents the posterior probability. https://doi.org/10.1371/journal.pone.0174366.g019 Syntermitinae. The soldier frontal-gland aperture is at the tip of a large projection located in the frontal region of the head; the soldier mandibles have a clearly recognizable molar plate and prominence, as in Silvestritermes [3], Cyrilliotermes [5], and Curvitermes [6]; and the worker gut morphology is very similar, including the characteristic dilated P1 of Syntermitinae [41]. Considering these points, the reallocation of the genus to Syntermitinae is expected, fol- lowing comprehensive studies of other Neotropical termitine genera. Defense and feeding behavior in Syntermitinae Two aspects stand out in termite research: defense and feeding habits. The first aspect relates to the soldier caste in Isoptera, which comprises a very particular case for evolutionary biology. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 20 / 29 Phylogenetic reconstruction of Syntermitinae Fig 20. Tree obtained with the Bayesian analysis with morphological data and COII, Cytb, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, and the branch color represents the posterior probability. https://doi.org/10.1371/journal.pone.0174366.g020 Soldiers are a ªburdenº on the colony maintenance, since they need to be fed by the workers, and the effective contribution of a very specialized caste for the colony defense is not clear. The proportion of soldiers and workers varies widely among species [42] and nearly 10% of termite species do not have soldiers (mainly Apicotermitinae). The second aspect relates to the central role of termites as decomposers in tropical climates; they can comprise as much as 95% of the soil insect biomass [43]. Termites can obtain nourishment from a variety of plant biomass sources, including wood, rotting wood, grass, cultures of fungi, lichen and humus; and this diversification of feeding habits appears to be linked to termite species diversification [21]. For the reconstruction of the defense behavior, each taxon was classified according to the categories of primary individual defense mechanisms summarized in [44]. Three categories of defense were recognized: ªBiting/Crushingº (example in Fig 3A) ªPiercingº (examples in Fig 3B and 3F), and ªSlashingº (examples in Fig 3C, 3D and 3E). Orthognathotermes sp. is formally PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 21 / 29 Phylogenetic reconstruction of Syntermitinae Fig 21. Comparison between topologies of the more consistent trees. A. analysis with morphological data and COII, 16S rDNA sequences, partitioned by genes; B. analysis with morphological data and all sequences, partitioned by codons; C. analysis with morphological data and COII, Cytb, 16S rDNA sequences, partitioned by codons. The equivalent branches are indicated by the colors; the name of each species is represented by an acronym. https://doi.org/10.1371/journal.pone.0174366.g021 classified as ªSlashing/Snappingº, but this is not relevant to the present discussion; the result is represented in Fig 22. The reconstruction showed that equivalent categories of defense evolved independently several times in syntermitine history: ªSlashingº mandibles appeared two or three times independently (Fig 22, black branches), ªPiercingº (Fig 22, blue branches) two or three times, and ªBiting/Crushingº (Fig 22, white branches) five or six times. Fig 22 shows two cases that well represent the degree of convergence of form. The soldier head of Rhynchotermes nasutissimus (Fig 22B) is clearly similar to Uncitermes teevani (Fig 22D), although U. teevani is more kindred to Labiotermes labralis (Fig 22C), which itself shares several traits with a distantly related species, Syntermes molestus (Fig 22A). These are not the only cases of convergence; the species of Embiratermes share a variety of external traits and are spread among four clades. It is unnecessary to exhaustively discuss all the cases, which would excessively lengthen this article. The soldier external morphology of each genus can be found in revisionary studies, or in the identification keys of Constantino [45, 46]. Other cases of convergence in termite soldier morphology were discussed by Inward and collaborators [21], who found that the asymmetrical snapping mandibles, a very specialized type of termite defense, evolved independently four times among all Isoptera. In the present case, we found a high degree of convergence in the soldier types of defense inside a much more restricted group. The means of establishing the diet of each termite species can be controversial. Some spe- cialists have proposed using analyses of the gut contents [47] or nitrogen stable-isotope ratios [48], but no discrete criteria have been developed to classify the termite diet precisely. Despite this, the resources consumed by termites can be organized in a continuous humification gradi- ent, from wood and grass, which are non-humified resources, at one extreme; and very humi- fied resources, such as humus and stercoral material from other nests, at the other [49]. This PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 22 / 29 Phylogenetic reconstruction of Syntermitinae Fig 22. Reconstruction of the primary individual defense mechanisms of syntermitine soldiers. Examples of soldier head shapes, A. Syntermes molestus; B. Rhynchotermes nasutissimus; C. Labiotermes labralis; D. Uncitermes teevani; the state of each taxon is indicated by the color of the squares, and the name of each species is represented by an acronym. https://doi.org/10.1371/journal.pone.0174366.g022 gradient can be correlated and recognized in the worker mandible morphology [50, 51]. Spe- cies that feed on non-humified resources have the molar region with conspicuous ridges and a relatively small apical tooth, which is termed ªxylophagous morphologyº (Fig 17A and 17B, for example). Species that feed on humified resources have the molar region without ridges and a prominent apical tooth, termed ªintermediate/geophagous morphologyº (Fig 17C±17E, for example). The reconstruction of these two characters (Fig 23), relative size of the left apical tooth (85) and the molar region (92), showed the expected overlap between these characteristics; xylopha- gous traits are traced in yellow and geophagous in black. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 23 / 29 Phylogenetic reconstruction of Syntermitinae Fig 23. Reconstruction of the syntermitine mandible characters. Molar region (character 92) and relative size of left apical tooth (character 85) reconstructions. The state of each taxon is indicated by the color of the squares, and the name of each species is represented by an acronym. https://doi.org/10.1371/journal.pone.0174366.g023 Our topology indicated that in the syntermitine evolutionary history, a very early split occurred between lineages that tend to feed on non-humified resources and species that tend to feed on very humified resources. The change in the species' diet was reflected in more than the mandible shape, and a complex change in the digestive apparatus and the associated sym- bionts would be expected; however, knowledge of Termitidae digestive processes and their correlation with the gut morphology is presently limited. We expect that the Syntermitinae will provide a useful and more practical case for future studies. Unfortunately, the lack of syntermitine fossils limits the dating and evolutionary interpreta- tions of these characteristics. The oldest record is an ichnofossil, described as a Syntermes-like nest [52], from southern Argentina and dating from the late Early Miocene; all other syntermi- tine fossil records in the literature are much more recent [53, 54]. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 24 / 29 Phylogenetic reconstruction of Syntermitinae Supporting information S1 Table. Character Matrix. (XLSX) S1 Fig. Traceplots of the analysis with morphological data and COII, 16S rDNA sequences, partitioned by genes. The two firsts plots correspond to each run and the low to the combined result. (TIF) S2 Fig. Traceplots of the analysis with morphological data and all four sequences, parti- tioned by codons. The two first plots correspond to each run and the low to the combined result. (TIF) S3 Fig. Traceplots of the analysis with morphological data and COII, Cytb, 16S rDNA sequences, partitioned by codons. The two first plots correspond to each run and the low to the combined result. (TIF) S4 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S5 Fig. Tree obtained with the Bayesian analysis with COII and 16S rDNA sequences, par- titioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S6 Fig. Tree obtained with the Bayesian analysis with COII and 16S rDNA sequences, par- titioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S7 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, COI, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S8 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, COI, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S9 Fig. Tree obtained with the Bayesian analysis with COII, COI and 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S10 Fig. Tree obtained with the Bayesian analysis with COII, COI and 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 25 / 29 Phylogenetic reconstruction of Syntermitinae S11 Fig. Tree obtained with the Bayesian analysis with morphological data and all four sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S12 Fig. Tree obtained with the Bayesian analysis with all four sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S13 Fig. Tree obtained with the Bayesian analysis with all four sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color rep- resents the posterior probability. (TIF) S14 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, Cyt b, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indi- cated above each node, the branch color represents the posterior probability. (TIF) S15 Fig. Tree obtained with the Bayesian analysis with COII, Cyt b, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S16 Fig. Tree obtained with the Bayesian analysis with COII, Cyt b, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S1 File. Additional information about the material used for DNA extractions in this work. (DOCX) Acknowledgments We thank Andrea P. V. Niño (U.D.C.A., Colombia), Cristian Dambros (INPA), Rudolf H. Scheffrahn (University of Florida) and Jan Sobotnõ Âk (Czech University of Life Sciences) for donating specimens; Jaqueline Battilana, Maria Augusta Ribeiro (MZUSP), Amanda F. Santos, Nara C. C. Pena Barbosa and Rullian Ce Âsar Ribeiro (FCAV-UNESP) for laboratory assistance and facilities; Tiago F. Carrijo, Joice P. Constantini and Rafaella G. Santos (MZUSP) for their help in field sampling and providing photographs; Olivia Evangelista, Kelli dos Santos Ramos, Tiago F. Carrijo and Rodolfo S. Probst (MZUSP) for help with the analyses and Janet W. Reid, for the careful English review. Author Contributions Conceptualization: MMR EMC. Data curation: MMR ACMC. Formal analysis: MMR ACMC. Funding acquisition: EMC. Investigation: MMR. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 26 / 29 Phylogenetic reconstruction of Syntermitinae Methodology: MMR CC. Project administration: MMR EMC. Resources: MMR EMC ACMC. Supervision: MMR EMC. Visualization: MMR. 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Phylogenetic reconstruction of Syntermitinae (Isoptera, Termitidae) based on morphological and molecular data

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Abstract

The subfamily Syntermitinae comprises a group of Neotropical termites with 18 genera and 101 species described. It has been considered a natural group, but relationships among OPENACCESS the genera within the subfamily remain uncertain, and some genera appear to be non-mono- Citation: Rocha MM, Morales-Corrêa e Castro AC, phyletic. Here, we provide a comprehensive phylogeny including six Neotropical species Cuezzo C, Cancello EM (2017) Phylogenetic reconstruction of Syntermitinae (Isoptera, of Termitinae as outgroup, 42 Syntermitinae species as ingroup, 92 morphological charac- Termitidae) based on morphological and ters (from external and internal anatomy of soldier and worker castes) and 117 molecular molecular data. PLoS ONE 12(3): e0174366. sequences (109 obtained for this study and 8 from GenBank) of 4 gene regions (41 and 22 https://doi.org/10.1371/journal.pone.0174366 from Cytochrome Oxidase I and II respectively, 19 from Cytochrome b, and 35 from 16S Editor: Tony Robillard, Museum National d'Histoire rDNA). Morphological and molecular data were analyzed in combination, with the Bayesian Naturelle, FRANCE inference method, and the important aspects of termite biology, defense and feeding habits Received: May 18, 2016 are discussed based on the resulting tree. Although useful for providing diagnostic charac- Accepted: March 8, 2017 ters, the morphology of the soldier caste reveals several cases of convergence; whereas Published: March 22, 2017 the feeding habit shows indications of evolutionary significance. Copyright:© 2017 Rocha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Introduction reproduction in any medium, provided the original author and source are credited. The subfamily Syntermitinae comprises a group of Neotropical termites that ranges from southern Mexico (Cahuallitermes) to northern Argentina (Cornitermes, Procornitermes, Data Availability Statement: All relevant data are within the paper and its Supporting Information Rhynchotermes, Syntermes), with the richest generic and specific diversity in the Brazilian Cer- files. rado biome. Fifteen syntermitine genera occur in the Cerrado, where several species of Corni- termes, Silvestritermes and Syntermes construct conspicuous epigeal nests that characterize this Funding: We received support from the São Paulo Research Foundation, Brazil (FAPESP) [http://www. savanna-like landscape. Cornitermes cumulans can reach a nest density of 55/ha, and is consid- fapesp.br/], grants 2012/00952-9 to MMR, 2013/ ered a keystone species in the Cerrado [1]. These termite nests may harbor many other termite 20068-9 to EMC and 2013/05610-1 to CC, and species as well as other groups of invertebrates. from the National Council for Scientific and The feeding and nesting habits of syntermitine species are diverse. The group includes Technological Development, Brazil (CNPq) [http:// grass/litter-feeders, intermediate feeders, and humus-feeders. The nests are variable; some spe- cnpq.br/], grant 308227/2013-0 to EMC. The funders had no role in study design, data collection cies build earthen nests; most are commonly epigeal, but arboreal and subterranean forms are PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 1 / 29 Phylogenetic reconstruction of Syntermitinae and analysis, decision to publish, or preparation of well known. Other nesting habits include inquilines, reformers, and diffuse galleries in the the manuscript. ground. A total of 18 genera and 101 species are now established as part of the subfamily. Some of Competing interests: The authors have declared that no competing interests exist. the taxa treated in taxonomic revisions and original descriptions in the last 20 years are Acan- gaobitermes [2], Armitermes [3], Cahuallitermes [4], Cyrilliotermes [5], Curvitermes [6], Labio- termes [7], Macuxitermes [8, 9], Noirotitermes [10], Paracurvitermes [11], Rhynchotermes [12], and Syntermes [13]. However, the status of Embiratermes is still in need of revision [3]. Engel and Krishna [14] proposed the subfamily, including only Cornitermes, Labiotermes, Procornitermes and Syntermes. Lately, Constantino and Carvalho [11] gave a new diagnosis, considering all the genera of ªmandibulate nasutesº then described (those genera with soldiers having developed mandibles and a recognizable frontal tube). Although Syntermitinae is a recently proposed taxonomic category, the ªmandibulate nasutesº group was recognized very early in the termite literature. The ªmandibulate nasutesº together with ªtrue nasutesº (group of genera with soldiers having vestigial mandibles and a developed frontal tube) were consid- ered part of the worldwide subfamily Nasutitermitinae. In the last century, two hypotheses were proposed regarding the origin of the nasute soldier: the monophyletic hypothesis, where the ªmandibulate soldiersº form an ancestral group of the ªtrue nasutesº [15, 16]; and the diphyletic hypothesis, where two independent lineages of ªmandibulate soldiersº led to the ªtrue nasutesº [17±20]. Inward and collaborators [21], in a comprehensive phylogenetic analysis with morphologi- cal and molecular data, supported the hypothesis that ªmandibulate nasutesº and ªtrue nasutesº are two distinct, independent lineages, and that the Syntermitinae is more closely related to the Amitermes-group (Termitinae) than to the Nasutitermitinae. Rocha and collaborators [3] developed a revisionary proposal for the genus Armitermes “sensu lato”, which included a cladistic analysis involving morphological characters from all species of Armitermes and representatives of all genera of ªmandibulate nasutesº. In this phylo- genetic approach, the genus Armitermes appears as polyphyletic, and some species are relo- cated to new genera, although the relationships among Syntermitinae genera are poorly resolved. Herein, we propose a comprehensive phylogenetic hypothesis for Syntermitinae, based on combined morphological and molecular data under a Bayesian approach; and reconstruct some aspects of the defense behavior and feeding habits of the group. Material and methods Taxon sampling and outgroup selection We included a total of 42 syntermitine species as ingroup, representing the diversity of the 18 currently described syntermitine genera; and 6 species of Termitinae as outgroup, chosen for their established relationships to Syntermitinae [21±23] and also based on our experience with Neotropical termites. Morphological studies were carried out on termite specimens deposited in the Isoptera collection of the Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil (MZUSP). A representative sample of each lot used to perform the molecular studies was formally deposited in the MZUSP as well and appropriately registered for public consult. Morphological characters We included a total of 92 characters, 40 of the soldier external morphology, 42 of the coiling gut in situ and the configuration of the different parts of the worker digestive tube, and 10 of worker external morphology. The morphological character data are expanded from our previ- ous study [3]; most characters are referenced in Figs 1±17. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 2 / 29 Phylogenetic reconstruction of Syntermitinae Fig 1. Examples of shapes of labrum. A. Syntermes molestus; B. Procornitermes triacifer; C. Microcerotermes strunckii; D. Labiotermes labralis; E. Cornitermes cumulans F. Silvestritermes holmgreni. https://doi.org/10.1371/journal.pone.0174366.g001 Fig 2. Examples of shapes of postmentum. A. Cornitermes cumulans; B. Labiotermes labralis; C. Rhynchotermes nasutissimus. https://doi.org/10.1371/journal.pone.0174366.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 3 / 29 Phylogenetic reconstruction of Syntermitinae Fig 3. Examples of types of frontal gland openings and frontal tube shapes. A. Microcerotermes strunckii; B. Amitermes amifer; C. Syntermes molestus; D. Labiotermes labralis; E. Procornitermes araujoi; F. Embiratermes festivellus. https://doi.org/10.1371/journal.pone.0174366.g003 The character matrix (S1 Table) was edited and managed with Mesquite v.3.04 [24]. Soldier head. 01. Labrum, hyaline tip: (0) absent (Fig 1C and 1F); (1) present (Fig 1A, 1B, 1D and 1E). PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 4 / 29 Phylogenetic reconstruction of Syntermitinae Fig 4. Examples of shapes of outer margins of the forecoxae and projections. A. Syntermes molestus; B. Cornitermes cumulans; C. Armitermes spininotus; D. Embiratermes festivellus; E. Rhynchotermes nasutissimus. https://doi.org/10.1371/journal.pone.0174366.g004 02. Shape of hyaline tip: (0) flat (Fig 1A and 1D); (1) fingerlike (Fig 1B and 1E). 03. Silhouette in dorsal view: (0) cuspidate (Fig 1A, 1B and 1E); (1) lanceolate (Fig 1C); (2) obtuse (Fig 1D and 1F). 04. Cuspidate margins: (0) slender (Fig 1A and 1E); (1) clearly angulate (Fig 1B). 05. Postmentum lateral margins: (0) angled (Fig 2A); (1) sinusoidal (Fig 2B); (2) convex (Fig 2C). 06. Postmentum length: (0) elongated (Fig 2A and 2B); (1) shorter (Fig 2C). 07. Shape of head in dorsal view: (0) rectangular, elongated; (1) rectangular, sort; (2) rounded. Fig 5. Examples of lateral lobes of the pronotum. A. Labiotermes labralis; B. Embiratermes festivellus; C. Syntermes molestus. https://doi.org/10.1371/journal.pone.0174366.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 5 / 29 Phylogenetic reconstruction of Syntermitinae Fig 6. Examples of shapes of thoracic nota. A. Labiotermes labralis; B. Armitermes spininotus; C. Syntermes molestus; D. Syntermes crassilabrum. https://doi.org/10.1371/journal.pone.0174366.g006 08. Number of antennal articles: (0) 20; (1) 19; (2) 18; (3) 17; (4) 16; (5) 15; (6) 14; (7); 13; (8); 12 (9) 11. 09. Head capsule microsculpture: (0) absent; (1) present (character 7 of [3]). 10. Visibility of frontal pore aperture: (0) indistinct (Fig 3A); (1) distinct (Fig 3B±3F). 11. Frontal pore shape: (0) retracted and narrow (Fig 3B); (1) protruded and wide (Fig 3C± 3F). 12. Membranous tissue at frontal pore aperture: (0) absent (Fig 3B and 3C); (1) present (Fig 3D±3F). Fig 7. Examples of mandibles (right) and molar regions. A. Procornitermes araujoi; B. Embiratermes festivellus; C. Curvitermes odontognathus. (Arrows indicate the molar region). https://doi.org/10.1371/journal.pone.0174366.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 6 / 29 Phylogenetic reconstruction of Syntermitinae Fig 8. Examples of pulvilli ornamentations. A. Uncitermes teevani; B. Mapinguaritermes peruanus. https://doi.org/10.1371/journal.pone.0174366.g008 13. Frontal projection: (0) absent (Fig 3A±3C); (1) present (Fig 3D and 3E). 14. Length of frontal tube: (0) reaching clypeus (Fig 3E); (1) surpassing clypeus (Fig 3F); (2) lump in profile (Fig 3D); (3) salience (Fig 3C). Soldier thorax. 15. Row of stout bristles on outer margins of forecoxae: (0) absent; (1) present (character 29 of [3]). 16. Stout bristles along femur: (0) absent; (1) present (as described for Labiotermes [7]). 17. Ornaments on internal face of tibia: (0) flat; (1) row of 20 spines. 18. Tibial spurs formula: (0) 3:2:2; (1) 2:2:2 19. Shape of outer margin of forecoxae: (0) nearly straight (Fig 4A); (1) with lump (Fig 4B). 20. Outer margin of forecoxae, distal portion: (0) round (Fig 4A and 4B); (1) keeled (Fig 4C); (2) spiniform (Fig 4D). 21. Spine on proximal portion of coxae: (0) absent (Fig 4A±4D); (1) present (Fig 4E). 22. Lateral lobes of pronotum: (0) not projected (Fig 5A); (1) slightly projected (Fig 5B); (2) well projected (Fig 5C). Fig 9. Insertion of the stomodeal valve in the mesenteron (A, B) and examples of alignment of the mesenteric tongues (C±F). A. Cornitermes cumulans; B. Procornitermes striatus; C. Silvestritermes holmgreni; D. Mapinguaritermes peruanus; E. Rhynchotermes nasutissimus; F. Ibitermes curupira. https://doi.org/10.1371/journal.pone.0174366.g009 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 7 / 29 Phylogenetic reconstruction of Syntermitinae Fig 10. Homology between the ornament regions inside the first proctodeal segment (red, central area; yellow, distal area; and green, region around the mesenteric tongue). A Curvitermes odontognathus; B. Embiratermes festivellus; C. Cornitermes cumulans. https://doi.org/10.1371/journal.pone.0174366.g010 23. Lateral margins of lobes (pronotum): (0) rounded (Fig 6A); (1) angulate (Fig 6C); (2) acuminate (Fig 6B and 6D). 24. Lateral margins of lobes (mesonotum): (0) rounded (Fig 6A); (1) angulate (Fig 6B and 6C); (2) acuminate (Fig 6D). 25. Lateral margins of lobes (metanotum): (0) rounded (Fig 6A and 6B); (1) angulate (Fig 6C); (2) acuminate (Fig 6D). 26. Outer margin of pronotum: (0) smooth (Fig 6A, 6C and 6D); (1) serrated (Fig 6B). 27. Outline of margins of metanotum and mesonotum: (0) smooth (Fig 6C and 6D); (1) ser- rated (Fig 6A and 6B). Soldier mandibles. 28. Molar region: (0) indistinct (Fig 7A); (1) distinct (Fig 7B and 7C). Fig 11. Examples of transition from tubular portion to dilated portion in P1 (arrows, initial portion of the dilated regions). A. Silvestritermes holmgreni; B. Cornitermes cumulans; C. Cyrilliotermes angulariceps; D. Uncitermes teevani. https://doi.org/10.1371/journal.pone.0174366.g011 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 8 / 29 Phylogenetic reconstruction of Syntermitinae Fig 12. Examples of connections between P1 and P3 through P2. A. Silvestritermes holmgreni; B. Cyrilliotermes angulariceps; C. Embiratermes festivellus. https://doi.org/10.1371/journal.pone.0174366.g012 29. When distinct, relative size of molar region: (0) reduced (Fig 7B); (1) developed (Fig 7C). 30. First marginal tooth of left mandible: (0) absent; (1) present 31. Second marginal tooth of left mandible: (0) absent; (1) present. 32. Shape between first and second marginal teeth of left mandible: (0) ªVº concavity; (1) cutting edge Fig 13. Examples of P2 insertion, relative to abdomen length (arrows, P2 position). A. Silvestritermes holmgreni; B. Cyrilliotermes angulariceps. https://doi.org/10.1371/journal.pone.0174366.g013 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 9 / 29 Phylogenetic reconstruction of Syntermitinae Fig 14. Examples of enteric valve shapes. A. Amitermes amifer; B. Curvitermes odontognathus; C. Mapinguaritermes peruanus; D. Genuotermes spinifer; E. Embiratermes festivellus; F. Embiratermes silvestrii; G. Procornitermes lespessi; H. Cornitermes cumulans; I. Silvestritermes holmgreni. https://doi.org/10.1371/journal.pone.0174366.g014 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 10 / 29 Phylogenetic reconstruction of Syntermitinae Fig 15. Examples of P3b shapes and isthmus insertions (arrow, sub-apical P3 insertion). A. Microcerotermes strunckii; B. Curvitermes odontognathus; C. Embiratermes ignotus; D. Acangaobitermes krishnai. https://doi.org/10.1371/journal.pone.0174366.g015 33. Shape of cutting edge between first and second marginal teeth of left mandible: (0) smooth; (1) serrated. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 11 / 29 Phylogenetic reconstruction of Syntermitinae Fig 16. Examples of body proportions and profiles. A. Cornitermes cumulans; B. Labiotermes labralis; C. Acangaobitermes krishnai. https://doi.org/10.1371/journal.pone.0174366.g016 34. First marginal tooth of right mandible: (0) absent; (1) present 35. Second marginal tooth of right mandible: (0) absent; (1) present. 36. Position of second marginal tooth: (0) in proximal portion; (1) in middle. 37. Shape of apical tooth: (0) slender; (1) strong; (2) sinuous. 38. Apical tooth curvature: (0) strongly arched; (1) slightly arched. 39. Internal outline of apical tooth: (0) concave (Fig 7B); (1) sinusoidal (Fig 7A). 40. Subapical tooth: (0) present; (1) absent. Characters of the gut anatomy. 41. Gizzard: Ornamentation of first-order pulvilli: (0) without notable ornaments (Fig 8A); (1) with developed spines (Fig 8B). 42. Insertion of stomodeal valve in mesenteron: (0) apical (Fig 9A); (1) subapical (Fig 9B). 43. Mesenteric tongue: (0) absent; (1) present. 44. Mesenteric tongue, proximal portion: (0) robust (Fig 9C, 9D and 9F); (1) constricted (Figs 9E and 11B); (2) filiform. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 12 / 29 Phylogenetic reconstruction of Syntermitinae Fig 17. Worker mandibles (not to same scale). A. Microcerotermes strunckii; B. Cornitermes cumulans; C. Silvestritermes holmgreni; D. Curvitermes odontognathus; E. Paracurvitermes manni; F. detail of C. cumulans molar plate notch; G. detail of S. holmgreni molar plate notch. https://doi.org/10.1371/journal.pone.0174366.g017 45. Alignment of mesenteric tongue: (0) continuous with external face of the mesenteric arch (Fig 9C); (1) with apex turned (Fig 9D); (2) laterally to mesenteric arch (Fig 9); (3) twisted (Fig 9F). 46. Secondary mesenteric tongue: (0) absent; (1) present. 47. Malpighian tubules attachment: (0) in two pairs; (1) four united. 48. Large ampulla at insertions of Malpighian tubules: (0) absent; (1) present. 49. Position of Malpighian tubules: (0) internal to mesenteric arch; (1) external to mesen- teric arch. Internal ornamentation of first proctodeal segment. The P1 internal ornaments were described by Rocha and Constantini [25], and the homology between the regions adopted in this study is explained in Fig 10 [central area in red, distal area in yellow, and around the mesenteric tongue(s) in green]. 50. Ornaments: (0) absent; (1) present. 51. Type of spines covering central area (0) Small spines in rows; (1) aciculiform; (2) robust spines; (3) as thin setae; (4) trifurcated spines. 52. Degree of sclerotization of spines: (0) slightly sclerotized; (1) strongly sclerotized. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 13 / 29 Phylogenetic reconstruction of Syntermitinae 53. Pattern of spine coverage in central area: (0) transverse; (1) longitudinal; (2) spaced. 54. Position of spines of central area, relative to mesenteric tongue: (0) after mesenteric ton- gue; (1) lateral to mesenteric tongue. 55. Central ridges: (0) absent; (1) present. 56. Spines around mesenteric tongue: (0) absent; (1) present. 57. Type of spines around mesenteric tongue: (0) single; (1) in small rows. 58. Coverage in distal area: (0) absent; (1) present. 59. Pattern of coverage in distal area: (0) sparse; (1) grouped in three areas. 60. When grouped in three areas: (0) complete columns; (1) incomplete columns; (2) rounded areas. First proctodeal segment. 61. Shape of P1: (0) tubular; (1) dilated. 62. Length of P1 relative to abdomen: (0) nearly the same (relatively outstretched in the abdomen); (1) longer (relatively coiled inside the abdomen). 63. Shape of dilated portion: (0) fusiform; (1) globose. 64. Transition from tubular portion to dilated portion of P1, see arrows: (0) distally con- stricted (Fig 11A); (1) gradual (Fig 11B); (2) proximally constricted (Fig 11C); (3) very strangu- lated (Fig 11D). 65. P1 orientation in relation to body axis: (0) parallel (Fig 11A); (1) diagonal (Fig 11B). 66. Shape of P1 final portion: (0) tubular (Fig 12A); (1) conical (Fig 12C); (2) tubular and narrow. 67. Shape of tubular P1 final portion: (0) arched; (1) straight. Second proctodeal segment. 68. Position of P2 insertion relative to abdomen length: (0) distally (Fig 13A); (1) at midlength (Fig 13B). 69. Position of P2 insertion in dorsal view: (0) on left side of body; (1) on right side of body. 70. Symmetry of enteric valve armature: (0) hexa-radial; (1) tri-radial; (2) asymmetric. 71. Category of ridges: (0) large pads (Fig 14A); (1) simple ridge (Fig 14B); (2) ridge slightly dilated at apex (Fig 14C); (3) finger-like (Fig 14D±14F); (4) lobate (Fig 14G and 14H); (5) bul- bous (Fig 14I). 72. Type of finger-like ridge: (0) protruded (Fig 14D); (1) oblong (Fig 14E); (2) conical (Fig 14F). 73. Type of lobate ridge: (0) slightly lobed (Fig 14G); (1) auricular (Fig 14H). 74. Proportions of lobate ridge: (0) equal; (1) unequal. 75. When slightly dilated at apex, length: (0) elongated; (1) short. 76. Ornaments: (0) absent; (1) present. 77. Ornament coverage: (0) triangular; (1) aciculiform. Third proctodeal segment. 78. Initial portion of P3: (0) directly connected (Fig 12); (1) bottleneck (Fig 12B); (2) well-developed enteric valve seating (Fig 12C). 79. Smooth diverticulum of P3a: (0) absent; (1) present. 80. P3b shape in dorsal view: (0) globose (Fig 15B); (1) arched (Fig 15C and 15D); (2) not protruded (Fig 15A). 81. Direction of P3b when arched: (0) turned forward (Fig 15D); (1) turned to right side of body (Fig 15C). 82. Insertion of isthmus: (0) apical (Fig 15A and 15B); (1) sub-apical (Fig 15C and 15D, arrow). Characters based on external morphology of workers. 83. Size proportion of head to thorax: (0) head much larger than thorax (Fig 16A); (1) head size similar to thorax (Fig 16B and 16C). 84. Body in profile: (0) slender (Fig 16C); (1) waisted (Fig 16A and 16B). PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 14 / 29 Phylogenetic reconstruction of Syntermitinae 85. Mandibles: Relative size of left apical tooth: (0) smaller than M1 (Fig 17B); (1) equal to M1 (Fig 17A and 17C); (2) more prominent than M1 (Fig 17D and 17E). 86. Edge of apical tooth: (0) straight (Fig 17A, 17B and 17C); (1) concave (Fig 17D and 17E). 87. M3 tooth on left mandible: (0) present, conspicuous (Fig 17A); (1) present, reduced (Fig 17B±17E). 88. M2 tooth on right mandible: (0) present, conspicuous (Fig 17A±17C); (1) present, reduced (Fig 17D); (2) absent (Fig 17E). 89. Relative position of right M2 tooth: (0) near middle of M1 and molar plate (Fig 17C); (1) near M1 tooth (Fig 17A and 17D); (2) near molar plate (Fig 17B); (3) fused to M1 (Fig 17E). 90. Right M2 posterior margin: (0) straight; (1) concave. 91. Molar plate notch: (0) absent; (1) present, 90 degrees (Fig 17F, arrow); (2) present, more than 90 degrees (Fig 17G, arrow). 92. Molar region: (0) with ridges; (1) with reduced ridges; (2) without ridges. Molecular protocols We chose four regions of the mitochondrial genome, Cytochrome Oxidase I and II (COI ~600 bp, COII ~660 bp), Cytochrome b (Cyt B ~340 bp) and 16S rDNA (~430 bp). The DNA was extracted preferentially from the head and thorax of a single soldier individual preserved in 95% ethanol (Table 1), with the set of reagents from the DNeasy Blood & Tissue Kits (Qiagen), supplemented with 20 mg/ml proteinase K, following the manufacturer's protocol. The homogenates were incubated at 55ÊC for 3 h. The gene fragments were then amplified by poly- merase chain reaction, PCR [26]. The primers and the amplification conditions are listed in Table 2. PCR was performed in 25 μL reactions (12.5 μL PCR master mix Promega1, 0.6 μM of each primer, 3.0 μL of total DNA, and 3.5 μL deionized water). The amplified PCR products were determined by gel electrophoresis on a 1% agarose gel diluted in TAE buffer (1X) (0.04 M Tris base, 0.02 M acetic acid, and 1 mM EDTA). This same buffer was also used in 1-h elec- trophoresis runs in an 8-V/cm length gel. All reaction products were purified with Wizard1 SV Gel and PCR Clean-Up System (Promega), following the manufacturer's protocols. Puri- fied PCR products were sequenced with the same primers used in the original PCR reactions and the BigDye1 Terminator v3.1 Cycle Sequencing Kit, under the same conditions of PCR. The sense and antisense sequences obtained from each amplicon were assembled, and a con- sensus sequence for each gene was generated with Geneious v.8.1.7 analysis tools [27]. The nucleotide sequences reported here were submitted to the GenBank database under the acces- sion numbers indicated in Table 1. Analyses The saturation of the molecular data was assessed with DAMBE v.6.0.48 [33] using the test of substitution saturation by [34, 35]. The saturation test showed little saturation, indicating that the data were suitable for phylogenetic analysis (Iss < ISSc; p < 0.05). To evaluate the most useful data set, we made several tests combining different sets of sequences (the four gene sequences, COII + 16S rDNA + COI, COII + 16S rDNA + Cytb and only COII + 16S rDNA), with and without the morphological data, and with the protein-cod- ing sequences partitioned either by genes or by the codon position. The results are summa- rized in the Table 3. The models for each DNA data partition were determined using the JModel Test 2 [36] and PartitionFinder v1.1.0 [37], for the morphological data partition the states were unordered. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 15 / 29 Phylogenetic reconstruction of Syntermitinae Table 1. List of GenBank accession codes for each gene. MZUSPLot no. COII COI Cytb 16S rDNA Termitinae Amitermes amifer 23727 KX247014 n.d. n.d. KX247076 Amitermes nordestinus 16373 KX247015 n.d. n.d. KX247077 Cylindrotermes parvignathus 23881 *DQ442113.1 n.d. KX247054 KX247075 Genuotermes spinifer 16354 KX247016 KY379279 n.d. KX247078 Microcerotermes sp. 21513 KX247013 n.d. n.d. KX247074 Orthognathotermes sp. 16233 * DQ442213 n.d. n.d. KX247073 Syntermitinae Acangaobitermes krishnai 13670 n.d. n.d. n.d. KX247081 Armitermes spininotus 24420 KX247034 n.d. KX247062 KX247092 Cahuallitermes intermedius 15463 KX247037 n.d. n.d. n.d. Cornitermes acignathus 24421 KX247039 KX247051 n.d. n.d. Cornitermes bequaerti 15970 KY379285 KY379284 KX247065 KX247097 Cornitermes bolivianus 20596 n.d. KX247052 KX247067 KY379286 Cornitermes cumulans 24423 ** EU253899 ** EU253860.1 KX247064 KX247096 Cornitermes ovatus 20617 KX247038 n.d. KX247066 KX247098 Cornitermes silvestrii 16232 KX247040 KY379283 n.d. KX247099 Curvitermes odontognathus 20705 KX247018 n.d. n.d. KX247080 Cyrilliotermes angulariceps 20709 KX247027 KY379282 KX247061 n.d. Embiratermes brevinasus 24424 KX247022 n.d. KX247057 KX247083 Embiratermes festivellus 24425 KX247026 KY379281 KX247060 KX247085 Embiratermes heteropterus 24427 KX247035 n.d. n.d. KX247093 Embiratermes ignotus 20810 KX247020 KX247048 KX247055 n.d. Embiratermes neotenicus 23830 KX247025 KX247050 KX247059 n.d. Embiratermes silvestrii 24428 KX247021 KY379280 KX247056 KX247082 Ibitermes curupira 24429 KX247029 n.d. n.d. KX247087 Labiotermes emersoni 16219 KX247030 KY379278 n.d. KX247088 Labiotermes labralis 14771 KX247031 n.d. n.d. KX247089 Labiotermes leptothrix 20997 KX247032 n.d. n.d. KX247090 Labiotermes orthocephalus 14829 KX247033 n.d. n.d. KX247091 Macuxitermes triceratops 16103 n.d. n.d. n.d. KX247095 Mapinguaritermes peruanus 14490 KX247028 n.d. n.d. KX247086 Noirotitermes noiroti 24430 KX247019 n.d. n.d. n.d. Paracurvitermes manni 21026 KX247017 KY379277 n.d. KX247079 Procornitermes araujoi 16315 ** EU253902 ** EU253862 n.d. n.d. Procornitermes lespesii 24431 KX247041 KX247053 KX247068 n.d. Procornitermes triacifer 24432 n.d. KY379276 KX247069 KX247100 Rhynchotermes nasutissimus 15981 KX247042 n.d. n.d. KX247101 Rhynchotermes perarmatus 24433 KX247043 KY379275 n.d. KX247102 Silvestritermes holmgreni 20549 KX247023 KX247049 KX247058 n.d. Silvestritermes minutus 20553 KX247024 KY379274 n.d. KX247084 Syntermes crassilabrum 21044 KX247046 KY379273 KX247071 KX247105 Syntermes grandis 16338 ** EU253903 ** EU253863 n.d. n.d. Syntermes molestus 21069 KX247045 KY379272 KX247070 KX247104 Syntermes parallelus 14753 KX247047 n.d. KX247072 KX247106 Syntermes spinosus 21155 KX247044 n.d. n.d. KX247103 (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 16 / 29 Phylogenetic reconstruction of Syntermitinae Table 1. (Continued ) MZUSPLot no. COII COI Cytb 16S rDNA Uncitermes teevani 20574 KX247036 n.d. KX247063 KX247094 Sequences obtained from other papers are indicated by the asterisks * [21] ** [23], n.d.: no data. https://doi.org/10.1371/journal.pone.0174366.t001 The Bayesian inference analyses were performed with MrBayes version 3.2.1 [38], in the CIPRES Science Gateway V. 3.3 [39]; in all analyses, four chains were run for 50 million gener- ations and sampled every thousand generations (two runs). In all cases the burn-in limitation was determined by visual inspection of the trace-plot and evaluation of the effective sample size value (ESS) of the combined runs, using Tracer v1.6 [40]. The burn-in of 1% was sufficient. Ancestral character states were reconstructed with the help of Mesquite v.3.04 [24], by the parsimony criterion. Results and discussion From the total of 48 species used in our analyses, we obtained DNA data from three or four different gene for 25 species, two different sequences for 16 species and only one sequence for 4 species (Table 1). Two taxa are represented only by morphological data. About one third of the sequences information is absent. Although the poverty of sequences may compromise the results, the majority of taxa share COII and 16s rDNA information (The information for COII sequences is absent only in four taxa and for 16S rDNA in 10), the major part of lacking infor- mation is concentrated in COI and Cytb sequences. Among the trees obtained, three of them present informative topology (with few poly- tomies) and high posterior probabilities (especially in the basal nodes); 1)The result of an anal- ysis with Morphology + COII + 16S rDNA, partitioned by genes (COII: GTR +I + G, 16S rDNA: GTR +G) represented in Fig 18 and Fig 20A (29 nodes more than 0.9, 2 nodes equal Table 2. Sequences of primers and PCR profiles used. Gene Primer Sequence (5'! 3') Reference COI F-LCO GGTCAACAAATCATAAAGATATTGG [28] R-HCO TAAACTTCAGGGTGACCAAAAAATCA [28] COII F-Leu TCTAATATGGCAGATTAGTGC [29] R-Lys GAGACCAGTACTTGCTTTCAGTCATC [29] Cyt B cytb612 CCATCCAACATCTCCGCATGATGAAA [30] cytb920 CCCTCAGAATGATATTTGGCCTCA [30] 16S rDNA 16SAr CGCCTGTTTATCAAAAACAT [31] 16SF TTACGCTGTTATCCCTAA [32] Conditions Gene Heat Denaturation Annealing Extension Final extension Cycles COI 94ÊC (2 min) 94ÊC (1 min) 43ÊC (1 min)* 72ÊC (1 min 15 s) 72ÊC (7 min) 40 COII 94ÊC (2 min) 94ÊC (1 min) 45 to 53ÊC (1 min)* 72ÊC (1 min 15 s) 72ÊC (7 min) 40 Cyt B 94ÊC (2 min) 94ÊC (1 min) 50ÊC (1 min) 72ÊC (1 min 15 s) 72ÊC (7 min) 40 16S rDNA 94ÊC (2 min) 94ÊC (1 min) 50ÊC (1 min) 72ÊC (1 min 15 s) 72ÊC (7 min) 40 *every 2ÊC, the temperature was maintained for 30 s. https://doi.org/10.1371/journal.pone.0174366.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 17 / 29 Phylogenetic reconstruction of Syntermitinae Table 3. Summarized results of combining different sets of sequences and types of codification for protein-coding sequences and their respective Estimated sample sizes (ESS) of each run combined. COII; COI; Cytb; 16S rDNA COII; COI; 16S rDNA COII; Cytb; 16S rDNA COII;16S rDNA Without the morphological data Protein-coding S13 Fig S10 Fig S16 Fig S6 Fig sequences partitioned by genes ESS: 60648 ESS: 63697 ESS: 56201 ESS: 50544 Without the morphological data Protein-coding S12 Fig S9 Fig S15 Fig S5 Fig sequences partitioned by the codon position ESS: 58695 ESS: 60012 ESS:54663 ESS:51510 With morphological data Protein-coding S11 Fig S8 Fig S14 Fig Fig 18 sequences partitioned by genes ESS: 56726 ESS: 56635 ESS: 60688 ESS: 65654 With morphological data Protein-coding Fig 19 S7 Fig Fig 20 S4 Fig sequences partitioned by the codon position ESS: 56656 ESS: 55645 ESS: 56361 ESS: 57063 https://doi.org/10.1371/journal.pone.0174366.t003 0.89, and 7 nodes less than 0.8); 2) The result of Morphology + all sequences, with protein-cod- ing genes partitioned by codons (1st codon: GTR+G, 2nd codon: HKY+I+G, 3rd codon: GTR +I+G, 16S rDNA: GTR+I+G), represented in Fig 19 and Fig 20B (26 nodes more than 0.9, 4 nodes between 0.8 and 0.9, and 10 nodes less than 0.8); 3) The result of Morphology COII + 16S rDNA + Cytb, with protein-coding genes partitioned by codons (1st codon: SYM+I+G, 2nd codon: HKY+I+G, 3rd codon: GTR+I+G, 16S rDNA: GTR+I+G), represented in Fig 20 and Fig 20C (30 nodes more than 0.9, 3 nodes between 0.8 and 0.9, and 9 nodes less than 0.8). Their respective traceplots are illustrated in S1±S3 Figs. Considering all obtained trees (Figs 18±20 and S4±S16 Figs), the exclusion of morphological data from the analysis result in large pectinate nodes (S5, S6, S9, S10, S12, S13, S15 and S16 Figs). The position of Acangaobitermes krishnai, Cahuallitermes intermedius, Macuxitermes tricer- atops and Noirotitermes noiroti represented by only one sequence and Armitermes armiger and Ibitermes tellustris, with no sequences, remains stable in the three more consistent trees (Fig 21), and considering just morphological characters, mainly from internal morphology, the position among them seems reliable. Comparing the three most robust results (Fig 21A±21C), their topologies have a few diver- gences; the groups of genera (delimited by the best-supported basal nodes, and indicated by the same colors in each tree) were recovered with identical taxa compositions. The more notable differences among the selected trees are: The position of the genus Syn- termes (the red branch, with taxa initiated by the acronym ªSYº), not resolved in tree A and positioned in trees B and C as a sister group of the yellow branch, composed of Rhynchotermes (indicated by the acronym ªRHº), Procornitermes (PR), Cahuallitermes (CA) and Cornitermes (CO). The relationships among the taxa are indicated by dark-green branches; Genuotermes (GESP), Curvitermes (CUOD), Embiratermes (EM), Paracurvitermes (PAMA), Cyrilliotermes (CYAN) and Silvestritermes (SI); possible paraphyletic in trees A and B, and recovered as a monophyletic group in tree C. Although these results do not contradict each other, for prudence we opted to reconstruct and discuss the ancestral character states using tree A, which is less resolved but more conservative. Taxonomic discussion The sample of species is sufficiently comprehensive to allow a discussion that previous studies did not attempt. Four genera appeared as paraphyletic in our analysis: Armitermes (possibly), Procornitermes (possibly), Embiratermes, and Ibitermes. The cases of Armitermes (pink branch, Fig 21) and Procornitermes (part of the yellow branch, Fig 21) can be resolved with reallocation of a few species: for Armitermes the most con- servative solution is include all taxons (Armitermes, Macuxitermes, Uncitermes and PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 18 / 29 Phylogenetic reconstruction of Syntermitinae Fig 18. Tree obtained with the Bayesian analysis with morphological data and COII, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, and the branch color represents the posterior probability. https://doi.org/10.1371/journal.pone.0174366.g018 Embiratermes heterotypus) into a single genus, since the node that it is grouping the five taxa has a high posterior probability. Nevertheless, considering we only obtain one sequences for Macuxitermes, and also the support for the three more internal nodes are low, we think more studies are necessary to infer consistently the relationship among the taxa before introducing nomenclatural changes; for Procornitermes, resurrecting it from Triacitermes Emerson, now including Procornitermes triacifer and P. araujoi is a admissible solution, however the para- phyly of the genus is not a consensus among the results. More detailed studies for these cases are necessary before formal proposals for nomenclatural changes can be made. A revisionary work is necessary in the next future to reassess generic and specific limits as well as the intergeneric relationships of Embiratermes and Ibitermes within other members of the subfamily, since their named species used herein as terminals are spread all over the tree. A surprising result is the position of Genuotermes, deeply inserted in our tree. Although the association with Syntermitinae is unintuitive, some unique characteristics are shared with PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 19 / 29 Phylogenetic reconstruction of Syntermitinae Fig 19. Tree obtained with the Bayesian analysis with morphological data and all four sequences, partitioned by codons. The respective posterior probability is indicated above each node, and the branch color represents the posterior probability. https://doi.org/10.1371/journal.pone.0174366.g019 Syntermitinae. The soldier frontal-gland aperture is at the tip of a large projection located in the frontal region of the head; the soldier mandibles have a clearly recognizable molar plate and prominence, as in Silvestritermes [3], Cyrilliotermes [5], and Curvitermes [6]; and the worker gut morphology is very similar, including the characteristic dilated P1 of Syntermitinae [41]. Considering these points, the reallocation of the genus to Syntermitinae is expected, fol- lowing comprehensive studies of other Neotropical termitine genera. Defense and feeding behavior in Syntermitinae Two aspects stand out in termite research: defense and feeding habits. The first aspect relates to the soldier caste in Isoptera, which comprises a very particular case for evolutionary biology. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 20 / 29 Phylogenetic reconstruction of Syntermitinae Fig 20. Tree obtained with the Bayesian analysis with morphological data and COII, Cytb, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, and the branch color represents the posterior probability. https://doi.org/10.1371/journal.pone.0174366.g020 Soldiers are a ªburdenº on the colony maintenance, since they need to be fed by the workers, and the effective contribution of a very specialized caste for the colony defense is not clear. The proportion of soldiers and workers varies widely among species [42] and nearly 10% of termite species do not have soldiers (mainly Apicotermitinae). The second aspect relates to the central role of termites as decomposers in tropical climates; they can comprise as much as 95% of the soil insect biomass [43]. Termites can obtain nourishment from a variety of plant biomass sources, including wood, rotting wood, grass, cultures of fungi, lichen and humus; and this diversification of feeding habits appears to be linked to termite species diversification [21]. For the reconstruction of the defense behavior, each taxon was classified according to the categories of primary individual defense mechanisms summarized in [44]. Three categories of defense were recognized: ªBiting/Crushingº (example in Fig 3A) ªPiercingº (examples in Fig 3B and 3F), and ªSlashingº (examples in Fig 3C, 3D and 3E). Orthognathotermes sp. is formally PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 21 / 29 Phylogenetic reconstruction of Syntermitinae Fig 21. Comparison between topologies of the more consistent trees. A. analysis with morphological data and COII, 16S rDNA sequences, partitioned by genes; B. analysis with morphological data and all sequences, partitioned by codons; C. analysis with morphological data and COII, Cytb, 16S rDNA sequences, partitioned by codons. The equivalent branches are indicated by the colors; the name of each species is represented by an acronym. https://doi.org/10.1371/journal.pone.0174366.g021 classified as ªSlashing/Snappingº, but this is not relevant to the present discussion; the result is represented in Fig 22. The reconstruction showed that equivalent categories of defense evolved independently several times in syntermitine history: ªSlashingº mandibles appeared two or three times independently (Fig 22, black branches), ªPiercingº (Fig 22, blue branches) two or three times, and ªBiting/Crushingº (Fig 22, white branches) five or six times. Fig 22 shows two cases that well represent the degree of convergence of form. The soldier head of Rhynchotermes nasutissimus (Fig 22B) is clearly similar to Uncitermes teevani (Fig 22D), although U. teevani is more kindred to Labiotermes labralis (Fig 22C), which itself shares several traits with a distantly related species, Syntermes molestus (Fig 22A). These are not the only cases of convergence; the species of Embiratermes share a variety of external traits and are spread among four clades. It is unnecessary to exhaustively discuss all the cases, which would excessively lengthen this article. The soldier external morphology of each genus can be found in revisionary studies, or in the identification keys of Constantino [45, 46]. Other cases of convergence in termite soldier morphology were discussed by Inward and collaborators [21], who found that the asymmetrical snapping mandibles, a very specialized type of termite defense, evolved independently four times among all Isoptera. In the present case, we found a high degree of convergence in the soldier types of defense inside a much more restricted group. The means of establishing the diet of each termite species can be controversial. Some spe- cialists have proposed using analyses of the gut contents [47] or nitrogen stable-isotope ratios [48], but no discrete criteria have been developed to classify the termite diet precisely. Despite this, the resources consumed by termites can be organized in a continuous humification gradi- ent, from wood and grass, which are non-humified resources, at one extreme; and very humi- fied resources, such as humus and stercoral material from other nests, at the other [49]. This PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 22 / 29 Phylogenetic reconstruction of Syntermitinae Fig 22. Reconstruction of the primary individual defense mechanisms of syntermitine soldiers. Examples of soldier head shapes, A. Syntermes molestus; B. Rhynchotermes nasutissimus; C. Labiotermes labralis; D. Uncitermes teevani; the state of each taxon is indicated by the color of the squares, and the name of each species is represented by an acronym. https://doi.org/10.1371/journal.pone.0174366.g022 gradient can be correlated and recognized in the worker mandible morphology [50, 51]. Spe- cies that feed on non-humified resources have the molar region with conspicuous ridges and a relatively small apical tooth, which is termed ªxylophagous morphologyº (Fig 17A and 17B, for example). Species that feed on humified resources have the molar region without ridges and a prominent apical tooth, termed ªintermediate/geophagous morphologyº (Fig 17C±17E, for example). The reconstruction of these two characters (Fig 23), relative size of the left apical tooth (85) and the molar region (92), showed the expected overlap between these characteristics; xylopha- gous traits are traced in yellow and geophagous in black. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 23 / 29 Phylogenetic reconstruction of Syntermitinae Fig 23. Reconstruction of the syntermitine mandible characters. Molar region (character 92) and relative size of left apical tooth (character 85) reconstructions. The state of each taxon is indicated by the color of the squares, and the name of each species is represented by an acronym. https://doi.org/10.1371/journal.pone.0174366.g023 Our topology indicated that in the syntermitine evolutionary history, a very early split occurred between lineages that tend to feed on non-humified resources and species that tend to feed on very humified resources. The change in the species' diet was reflected in more than the mandible shape, and a complex change in the digestive apparatus and the associated sym- bionts would be expected; however, knowledge of Termitidae digestive processes and their correlation with the gut morphology is presently limited. We expect that the Syntermitinae will provide a useful and more practical case for future studies. Unfortunately, the lack of syntermitine fossils limits the dating and evolutionary interpreta- tions of these characteristics. The oldest record is an ichnofossil, described as a Syntermes-like nest [52], from southern Argentina and dating from the late Early Miocene; all other syntermi- tine fossil records in the literature are much more recent [53, 54]. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 24 / 29 Phylogenetic reconstruction of Syntermitinae Supporting information S1 Table. Character Matrix. (XLSX) S1 Fig. Traceplots of the analysis with morphological data and COII, 16S rDNA sequences, partitioned by genes. The two firsts plots correspond to each run and the low to the combined result. (TIF) S2 Fig. Traceplots of the analysis with morphological data and all four sequences, parti- tioned by codons. The two first plots correspond to each run and the low to the combined result. (TIF) S3 Fig. Traceplots of the analysis with morphological data and COII, Cytb, 16S rDNA sequences, partitioned by codons. The two first plots correspond to each run and the low to the combined result. (TIF) S4 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S5 Fig. Tree obtained with the Bayesian analysis with COII and 16S rDNA sequences, par- titioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S6 Fig. Tree obtained with the Bayesian analysis with COII and 16S rDNA sequences, par- titioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S7 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, COI, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S8 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, COI, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S9 Fig. Tree obtained with the Bayesian analysis with COII, COI and 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S10 Fig. Tree obtained with the Bayesian analysis with COII, COI and 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 25 / 29 Phylogenetic reconstruction of Syntermitinae S11 Fig. Tree obtained with the Bayesian analysis with morphological data and all four sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S12 Fig. Tree obtained with the Bayesian analysis with all four sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S13 Fig. Tree obtained with the Bayesian analysis with all four sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color rep- resents the posterior probability. (TIF) S14 Fig. Tree obtained with the Bayesian analysis with morphological data and COII, Cyt b, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indi- cated above each node, the branch color represents the posterior probability. (TIF) S15 Fig. Tree obtained with the Bayesian analysis with COII, Cyt b, 16S rDNA sequences, partitioned by codons. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S16 Fig. Tree obtained with the Bayesian analysis with COII, Cyt b, 16S rDNA sequences, partitioned by genes. The respective posterior probability is indicated above each node, the branch color represents the posterior probability. (TIF) S1 File. Additional information about the material used for DNA extractions in this work. (DOCX) Acknowledgments We thank Andrea P. V. Niño (U.D.C.A., Colombia), Cristian Dambros (INPA), Rudolf H. Scheffrahn (University of Florida) and Jan Sobotnõ Âk (Czech University of Life Sciences) for donating specimens; Jaqueline Battilana, Maria Augusta Ribeiro (MZUSP), Amanda F. Santos, Nara C. C. Pena Barbosa and Rullian Ce Âsar Ribeiro (FCAV-UNESP) for laboratory assistance and facilities; Tiago F. Carrijo, Joice P. Constantini and Rafaella G. Santos (MZUSP) for their help in field sampling and providing photographs; Olivia Evangelista, Kelli dos Santos Ramos, Tiago F. Carrijo and Rodolfo S. Probst (MZUSP) for help with the analyses and Janet W. Reid, for the careful English review. Author Contributions Conceptualization: MMR EMC. Data curation: MMR ACMC. Formal analysis: MMR ACMC. Funding acquisition: EMC. Investigation: MMR. PLOS ONE | https://doi.org/10.1371/journal.pone.0174366 March 22, 2017 26 / 29 Phylogenetic reconstruction of Syntermitinae Methodology: MMR CC. Project administration: MMR EMC. Resources: MMR EMC ACMC. Supervision: MMR EMC. Visualization: MMR. 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