Proneotermes macondianus, a new drywood termite from Colombia and expanded distribution of Proneotermes in the Neotropics (Isoptera, Kalotermitidae)

Abstract After more than one hundred years, a new drywood termite of the genus Proneotermes is described from the tropical dry forest in the Caribbean coast of Colombia. Morphological and genetic analyses are given for Proneotermes macondianus sp. n. This termite occurs in tropical dry forests in small colonies inside thin branches of dry wood. The soldier of Proneotermes macondianus is smaller and the genal horns are angled outward compared to the other two described Proneotermes species. The imago wings are unusually short and wide. Genetic analyses for COII, 12S, and 16S genes show less than three percent difference between sample localities of Proneotermes macondianus. Intergeneric comparison with selected kalotermitid genera indicates that Bifiditermes is the most closely related genus of those sequenced. New morphological descriptions and morphometric measurements of Proneotermes latifrons based on the soldier caste are also included. Neotropical locality records for Proneotermes latifrons and Proneotermes perezi are provided.

For more than a century, the genus Proneotermes was represented by two species, Proneotermes latifrons (Silvestri, 1901) from Venezuela and Proneotermes perezi (Holmgren 1911) from Costa Rica (Krishna et al. 2013). DNA barcoding is a molecular tool used to identify and to track the evolutionary biology of species (Thompson et al. 2000, Inward et al. 2007, Hausberger et al. 2011, Bourguignon et al. 2014. Evolutionary analyses within the Kalotermitidae are incomplete and limited to some genera (Legendre et al. 2008). Hence a comparative genetic analysis can help to determine relationships for Proneotermes.
In this paper, a new species of Proneotermes is described, P. macondianus. In addition, new morphological descriptions are included for the soldier of P. latifrons and new soldier measurements provided for P. perezi as well as new locality records for Proneotermes in the Neotropics.

Study sites and sampling
Three study sites in a tropical dry forest near Colombia's Caribbean coast were selected and surveyed during July 2014 and August 2015 (Fig. 1). The area of "Los Primates" in the mountains of municipality of Colosó, Sucre and the "El Ceibal" in Santa Catalina Bolívar, are part of the system of protected areas, while the "El Parque Tayrona" is a Natural National Park of Colombia in Santa Marta, Magdalena. These forests are one of the best preserved areas of tropical dry forest in the Colombian Caribbean coast (Instituto de Investigación Alexander von Humboldt 2014). Samples of a new Proneotermes were collected in those places using a standardized sampling protocol (Jones andEggleton 2000, Hausberger andKorb 2015) that included collecting small dry branches and dry wood on the ground. Specimens were preserved in 100% ethanol for DNA analysis and 80% ethanol for museum curation.

Identification
Morphometrics for P. latifrons and P. perezi were obtained from specimens from the University of Florida Termite Collection, Davie, Florida. Specimens of P. macondianus sp. n. were also sequenced for genetic comparisons. Total DNA was extracted from pseudergates and alate imagoes heads using the CTAB protocol (Doyle and Doyle 1987). PCRs and sequencing were performed for mitochondrial gene fragments from cytochrome oxidase II (COII) (~740 bp), 12S rDNA (~385 bp), and 16S rDNA (~480 bp) as described in Hausberger et al. (2011).
For the three different haplotypes of P. macondianus from the northern Colombian coast (separated ~200 km from each other), we used the combined COII, 12S, and 16S nucleotide sequences to calculate the p-distance (3000 th Bootstrap replications, Gamma Distributed and Transitions + Transversions).
Due to limited availability of mitochondrial gene sequences for Kalotermitidae in National Center for Biotechnology Information (NCBI), we restricted our phylogenetic analysis to only the COII fragment. Twelve genera of Kalotermitidae and Cryptocercus punctulatus as the outgroup were used (Table 1). Sequences were aligned with MUSCLE alignment algorithm as implemented in MEGA 7.0 with default settings (Kumar et al. 2016). A phylogenetic tree was inferred based in a Bayesian approach using MrBayes 3.2.1. (Ronquist and Huelsenbeck and 2003) (10 7 generations with every 1000 th tree sampled, using the default of four chains). After checking for convergence, we discarded 50% as burn-in. The resultant tree was visualized using FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). Additionally, MEGA 7.0 was also employed (Kumar et al. 2016) to calculate p-distances (as described above) between all species using the COII fragment. All positions containing gaps and missing data were eliminated.

Imaging and measurements
Specimens were suspended in Hand Sanitizer and images were taken with a Leica MC205 C stereomicroscope coupled to a Leica MC190 HD digital camera. The software Helicon Focus was used to stack pictures. Measurements were done following Roonwal (1969). Wings and mandibles were detached and mounted onto slides and edited with Photoshop CS5 V12.0.

Deposit
Voucher specimens are held at the University of Freiburg, Germany. The holotype soldier and paratypes of Proneotermes macondianus will be deposited at the Natural History Museum of the Alexander von Humboldt Institute of Bogotá (MIAvH) and a paratype soldier at the collection of the American Museum of Natural History, New York. De-alates (wings detached) and pseudergates of P. macondianus will be part of the collection of the Department of Chemistry and Biology at the Universidad del Norte, Barranquilla, Colombia.

Family Kalotermitidae Froggatt, 1897 Genus Proneotermes Holmgren, 1911
Proneotermes macondianus sp. n. http: Diagnosis. The Proneotermes macondianus soldier is smaller and the head capsule lighter than those of P. latifrons and P. perezi. In P. macondianus, the lateral margins of the genal horns angle outward from the sides of the head capsule while, in the other two species, the lateral margins of the genal horns remain in line with the head capsule. The mandibular humps of P. macondianus are more pronounced and rounded than in P. latifrons and P. perezi. Both P. latifrons and P. perezi have more robust rugosity on the frons than P. macondianus. The imago of P. macondianus is smaller and has much shorter, wider, darker, and more punctate wings than that of P. perezi. Description. Imago (Figs 2, 3A, Table 2). Head dorsal view: yellowish weakly trapezoidal, eyes moderately protruding and small, diameter 0.30 mm ( Fig. 2A-B). Ocellus oval and almost touching eye (Fig. 2B). Antenna with 15 articles. Pronotum broader than head ( Fig. 2A). Forewing with all major veins running parallel; subcosta running from suture to costal margin about 1/5 length of wing, radius to 1/3 wing length, radial sector with 4-6 branches, media less sclerotized than anterior veins, and cubitus unsclerotized Wings brownish, especially near scale suture, membrane nodular; unusually wide and relatively short. Fore wing with a very long suture line margin; scale much darker that body pigmentation (Fig. 3A). Measurements are reported in Table 2.
Soldier (Figs 3B, 4, Table 3). Head in dorsal view with postclypeus almost black, grading to ferruginous orange near frontal flange, and yellow at occiput (Fig. 4A). Dorsal view with head elongate and sides parallel, frons wide and shallow and faint frontal flange (Fig. 4B). Eye spots distinct, unpigmented. Mandibles completely black (Fig. 3B, 4A-C). Pronotum yellowish with anterior borders brown. Frons angles below vertex approx. 43°. Rugosity vestigial on the frons or vertex regions of the head. Frontal horns robust and project towards the front (Fig. 4A). Genal horns prominent in dorsal view, angled antero-laterally about 45°. Mandible tips bend about 60-65° from longitudinal axis of mandibular blade, prominent dentition, with rounded and pronounced mandibular humps: left hump larger than right (Figs 3B, 4A). Postmentum somewhat constricted in middle, as cup-shaped (Fig. 4C). Third antennal article enlarged and sclerotized, formula 2<3>4=5=6 and 11 articles. Pronotum as broad as head; anterior emarginate. Measurements are reported in Table 3. The soldiers from the Tayrona National Park (Santa Marta, Magdalena) showed slightly darker coloration than those from the samples sites at El Ceibal (Santa Catalina, Bolívar) and Colosó (Colosó, Sucre). Genetic analysis. The COII, 12S, and 16S sequences obtained in this study are deposited in GenBank under accession numbers KX267090-KX267098 (Table 1). The combined COII, 12S, and 16S nucleotide data of three different haplotypes of P. macondianus from the northern Colombian coast (separated ~200 km from each other) revealed genetic distances of about 2.5% (p-distance, SE 0.004; 38bp / 1488bp)   (Table 4). Our Bayesian phylogenetic tree shows more than 74% Bayesian Posterior Probability (BPP) support for all nodes (Fig. 5)   Ecological notes. Proneotermes macondianus sp. n was found in tropical dry forests of the Colombian Caribbean near to coastal areas up to 25 km inland (Fig. 1, Appendix 1 - Figure S1). Encounters of Proneotermes were scarce. In line transects that covered a total area of 1500m × 2m, only 0.82% of all termite samples (n = 1102) were P. macondianus (n = 9). All samples were from thin pieces of drywood branches: less than 2 cm diameter on the ground, with a maximum of 20 individuals per branch. Pellets were hexagonal in shape, beige in colour and had a length of 0.92 +/-0.04 mm (Appendix 1: Figure S1, S2, Table S1). It was impossible to identify the plant species from the small dry branches where P. macondianus sp. n. was found.
Material examined. Holotype colony: Colombia: Municipality of Santa Marta, Magdalena. Tayrona National Natural Park, Gairaca Bay: 11.3152°N, 74.1032°W (Fig. 1), 6m, 27.VI.15 by R. Casalla. COLPT4K1-206. Holotype: Soldier, paratypes: 5 soldiers, 2 reproductives, and few pseudergates, two used for DNA analysis. Municipality of Santa Catalina, Bolívar. Protected area "El Ceibal": 10.6336°N, 75.2517°W, 25m, 30.VIII.14 R. Casalla. COLCE3F5-155, COLCE3G5-158, COLCE3H2-160: Paratypes: 4 soldiers, 5 functional reproductives, and few pseudergates, two used for DNA analysis. Municipality of Colosó, Sucre. Serranía de Coraza y Montes de María. Table 4. Nucleotide distances for combined analysis of COII, 12S rDNA and 16S rDNA genes between localities of P. macondianus sp. n. (p-distance). Standard error estimates are shown above the diagonal.   Protected área "Los Primates": 9.5332°N, 75.3479°W, 223m, 27.VII.14 R. Casalla. COLCO4F4-226: Paratypes: 1 soldier, 1 winged imago, 2 dealated imagoes, one used for DNA analysis. Measurements for holoype, paratype soldiers and imagoes are reported in Table 2 and 3. The holotype and clearly colored paratype soldiers from COLCE3F5-155 will be deposited in the Arthropod Collection of the Natural History Museum of the Alexander von Humboldt Institute of Bogotá, Colombia (MIAvH). A paratype soldier from holotype colony, will be deposited in the American Museum of Natural History, New York, United States. Morphotype imagoes, paratype soldiers and pseudergates will be part of the collection of the Department of Chemistry and Biology at the University del Norte, Barranquilla, Colombia. Etymology. Macondianus: In honour of Nobel laureate Gabriel García Marquez and the fictional town "Macondo" in his novel "One hundred years of solitude". "Macondiano/a" is also a Spanish world used in Colombia to describe an incredible, rare or surprising event that could only be compared with the fictional universe and magical realism of this novel. Silvestri's (1901Silvestri's ( , 1903 descriptions of the P. latifrons soldier are incomplete. Some characters such as frons angle, horns, and postmentum morphology were not included. Also morphometrical measurements are incomplete. Herein, we included morphometrical measurements for the soldier caste. The imago caste is unknown. Soldier (Fig. 4D-F, Table 6). Head in dorsal view with frons dark glossy until faint bridge, grading from ferruginous orange to orange-yellow toward vertex. Postclypeus whitish at borders. Mandibles black anteriorly and reddish brown at hump. Head in lateral view with dark ferruginous orange, then turns orange to genal region. Head in ventral view with postmentum chestnut-dark brown and whitish at anterior border and genal margin pale orange. Eye spots distinct, unpigmented. Pronotum hyaline with sclerotized borders. First three antennal segments darker.

Proneotermes latifrons
Head subsquare with sides slightly convergent, posteriorly and rounded to vertex. Frontal area wide and long, occupies ca 2/5 of head length to postclypeus; narrowly depressed in center, and laterally view with faintly convex and few undulations, sloping angle ca. 50° near to postclypeus. Labrum short and sub-squared. Antennal socket protruded with third antennal segment longer and sclerotized, formula 2<3>4=5=6 and 11 articulations. Postmentum very broad in front. Pronotum as broad as head with anterior emarginated. Mandibles strong and curved inward ca. 45-50°. Measurements are reported in Table 6.
Comparisons. Soldiers of P. latifrons are separated from congeners in having a wide and darker convex frons with narrow undulations dorso-laterally. Postclypeus whitish at border, labrum wider than long and darker postmentum. P. latifrons is distributed in Venezuela, while P. perezi is widely distributed in Central America, from Guatemala to Panama (Fig. 1). Smaller species; maximum head width 1.14-1.32 mm (mean 1.23 SD 0.06 mm). Lateral margins of the genal horns angle outward from the sides of the head capsule (Fig. 4A, C)  In lateral view; frons forms even curve below vertex and mandibles (Fig. 4E); postmentum about twice as long as wide. Posterior margin conical (Fig. 4F)  In lateral view; frons forms rather straight angle from vertex to mandibles (Fig. 4H); postmentum about three fifths as long as wide. Posterior margin convex (Fig. 4I)

Discussion
The phylogenetic relationships within the Kalotermitidae are not clearly resolved yet. Krishna (1961) hypothesized that, based on wing venation and morphology of imago mandibles, Proneotermes is sister group to a clade composed of Tauritermes, Allotermes, Mariginitermes, and Incisitermes. In contrast, our phylogenetic tree shows Proneotermes, a Neotropical group, is monophyletic (87% BPP), separated from ancestral line of those who originated Marginitermes and the Old World genus Bifiditermes, Epicalotermes, and New World Incisitermes and the Pantropical Cryptotermes. However, our results suggest that the genetic distances between Proneotermes and congeners are quite high (p-distance 0.153 -0.211 for COII fragment) and the closest genus to Proneotermes is Bifiditermes (Table 4). Using a single mitochondrial marker (COII) available for 12 Kalotermitidae genera, our results resemble those from Legendre et al. (2008), who included seven gene fragments in combination with morphological characters in their analyses. To fully resolve phylogenetic relationships within the cosmopolitan Kalotermitidae, a denser taxon sampling along with covering more genetic markers, ideally including nuclear loci and moreover morphological characters is needed.