A peer-reviewed open-access journal Zookeys 748: 21—30 (2018) doi: 10.3897/zookeys.748.24253 RESEARCH ARTICLE Z00Ke y S http:/ /Z00 keys -pen soft.net Launched to accelerate biodiversity research Echinotermes biriba, a new genus and species of soldierless termite from the Colombian and Peruvian Amazon (Termitidae, Apicotermitinae) Daniel Castro', Rudolf H. Scheffrahn’, Tiago F. Carrijo? | Instituto Amazénico de Investigaciones Cientificas SINCHI, Avenida Vasquez Cobo Calles 15 y 16, Leticia, Amazonas, Colombia 2. Fort Lauderdale Research and Education Center, Institute for Food and Agricultural Sciences, University of Florida, 3205 College Avenue, Davie, Florida 33314, USA 3 Centro de Ciéncias Natu- rais e Humanas, Universidade Federal do ABC, Rua Arcturus 03, Jardim Antares, 09606-070, Sao Bernardo do Campo, SP. Brazil Corresponding author: Daniel Castro (danielkaz80@gmail.com) Academic editor: D. Evangelista | Received 5 February 2018 | Accepted 14 March 2018 | Published 4 April 2018 /ttp://zoobank.org/27364E47-1566-48B0-8757-OB5 E32A4C0AA Citation: Castro D, Scheffrahn RH, Carrijo TF (2018) Echinotermes biriba, a new genus and species of soldierless termite from the Colombian and Peruvian Amazon (Termitidae, Apicotermitinae). ZooKeys 748: 21-30. https://doi. org/10.3897/zookeys.748.24253 Abstract A new Apicotermitinae genus and species Echinotermes biriba is described from workers collected on the Andean-Amazon Piedmont in Colombia and Peru. The enteric valve armature of Echinotermes biriba Castro & Scheffrahn, gen. et sp. n. is a remarkably diagnostic character. A Bayesian phylogenetic analysis using the COI gene and including all other Neotropical Apicotermitinae genera, supports the new genus as a distinct terminal. Keywords Anoplotermes-group, enteric valve, Neotropic, taxonomy Introduction The soldierless termites of Amazonia form a dominant group and comprise more than 30% of the termite diversity in neotropical assemblages (Davies 2002, Ackerman et al. 2009, Palin et al. 2011). Although the richness of soldierless taxa is recognized, Copyright Daniel Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Daniel Castro et al. / ZooKeys 748: 21-30 (2018) most have not been described yet (Bourguignon et al. 2015). For example, Palin et al. (2011) list four undescribed Anoplotermes species and 18 undescribed species in 13 undescribed genera from Peru. Originally, all neotropical soldierless termites were placed in the genus Azoplotermes Miller, 1873. Recognition of much greater taxo- nomic diversity began with Mathews (1977) who described Grigiotermes and Rupti- termes, and Fontes (1986) who described Aparatermes and Tetimatermes. Fontes (1992) provided the first identification key for workers of these five genera. The descriptions of Longustitermes (Bourguignon et al. 2010), Compositermes (Scheffrahn 2013), Amplu- crutermes, Humutermes, Hydrecotermes, Patawatermes, and Rubeotermes (Bourguignon et al. 2016), and Disjunctitermes (Scheffrahn et al. 2017) have advanced the classifica- tion of neotropical soldierless taxa but many more remain to be described. Currently, 13 genera and 52 species of Apicotermitinae are known from the Neo- tropical region (Bourguignon et al. 2010; Krishna et al. 2013; Scheffrahn 2013; Car- rijo et al. 2015; Bourguignon et al. 2016; Scheffrahn et al. 2017). For Colombia, Anoplotermes ater, Anoplotermes parvus, Aparatermes silvestrii, Humutermes krishnai, and Patawatermes turricola have been reported (Araujo 1977; Constantino 1998; Bour- guignon et al. 2016; Pinzén et al. 2017), and Peru records include Anoplotermes banksi, Anoplotermes pacificus, Disjunctitermes insularis, Rubeotermes jheringi, and Ruptitermes reconditus (Constantino 1998, Bourguignon et al. 2010, Acioli and Constantino 2015, Bourguignon et al. 2016, Scheffrahn et al. 2017). Only 19% of the species of Apico- termitinae of the Neotropics are reported in these two countries. In this paper Echinotermes biriba gen. n. et sp. n. is described based on the mor- phology of the worker caste and molecular data. Materials and methods The specimens were collected and preserved in 75% or 85% ethanol. ‘The dissection of the enteric valve (EV) was done by removing the P2 tube from the worker’s gut and then expelling all the food particles by means of controlled pressure. The tube was immersed in a PVA medium to completely detach the EV from surrounding muscle tissue and cut longitudinally to splay open the EV for mounting in the medium. The mandibles were also submerged in PVA medium. The terminology used for the worker gut follows Sands (1972) and Noirot (2001). The COI sequence of EF. biriba was obtained by DNA extraction and PCR per- formed by the Canadian Centre for DNA Barcoding following standard high-through- put protocols (deWaard et al. 2008). The PCR employed the primers LepF1 and LepR1 (Hebert et al. 2003) which generated 622 to 652bp of the barcode region of the mitochondrial gene cytochrome c oxidase subunit 1 (COI). A gene tree was created under Bayesian Inference (BI) using the COI gene. In ad- dition to the sequence of E. biriba, a total of 48 GenBank sequences were used: 34 se- quences of neotropical Apicotermitinae (21 species, 13 genera), eight non neotropical Apicotermitinae genera, five non-apicotermitine Termitidae, and one Rhinotermiti- Echinotermes biriba, a new genus and species of soldierless termite from the Colombian... 23 dae, (Heterotermes crinitus) as the outgroup. Sequences were aligned under MUSCLE algorithm implemented in Geneious v6.1.6 (Biomatters Ltd., Auckland, New Zea- land). Substitution model used (GTR+1+G) was selected through the Akaike Informa- tion Criterion (AIC) with the software jModelTest2 (Darriba et al. 2012). The XML input files were generated with BEAUti 1.8.0, and the BI was performed with BEAST 1.8.0 (Drummond et al. 2012). A Yule speciation process, with a random starting tree, and relaxed molecular clock was used as tree priors. Four Markov chain Monte Carlo (MCMC) searches were conducted, each one for 15,000,000 generations, and they were combined to search the most probable tree. Convergence and stationarity were assessed with Tracer 1.5 (Rambaut et al. 2014) and the first 600 trees were discarded as burn-in with TreeAnnotator 1.8.0 and visualized using FigTree 1.3.1. Systematics Echinotermes Castro & Scheffrahn, gen. n. http://zoobank.org/9872DC61-CA8C-42B5-9ABE-62160F532ECD Type-species. Echinotermes biriba sp. n. Imago. Unknown. Description of worker. (Fig. 1). Monomorphic. Head capsule and antennae a light yellowish colour; pronotum pale yellow; legs hyaline. Head covered with approx. 30 longer setae (0.1 mm) and approx. 100 shorter setae (< 0.05 mm) (Fig. 1A). In lat- eral view, dorsal surface of the head capsule slightly convex; postclypeus is moderately inflated. Antennae with 14 articles. Pronotum with four or five long setae and numer- ous short hairs. Mandibles with apical teeth more prominent than first marginal teeth; left mandible with M1+2 equilateral, M3 forming right angle, molar prominence pro- jecting in line with apical tooth; right mandible with concave margin between M1 and M2 (Fig. 1B). Fore-tibia moderately inflated (Fig. 1C) and covered with approx. 60 longer setae and approx. 40 shorter setae; pilosity denser apically. Third (external) spur very small. Femur with approx. 20 sparse large setae. Tibial spurs 2:2:2. Digestive tube (Fig. 1D) with very large crop, more voluminous than paunch (P3). Mesenteron forming complete 360° loop. Mesenteric tongue short, truncate. First proc- todeal segment tubular, equal diameter throughout and visible its entire length in ventral view. Enteric valve seating trilobed, with smaller lobe not visible in intact gut. Enteric valve with six cushions, terminating at the opening to the P3 as spiny spheroids (Fig. 2). Diagnosis. The crop of E. biriba is unusually large and the enteric valve armature, consisting of six spherical pectinate pads, is unique among all apicotermitine genera. Remarks. Mandibles of Rubeotermes jheringi and Humutermes krishnai are very similar to E. biriba, but the first marginal teeth of EF. biriba are less prominent that those two genera. The diagnostic character of FE. biriba is the enteric valve armature which is also spiked in the Humutermes enteric valve (EV) but in E. biriba the EV Daniel Castro et al. / ZooKeys 748: 21-30 (2018) Figure |. Worker of Echinotermes biriba sp. n.: A dorsal and lateral views of head and pronotum B mandibles C right fore-tibia D digestive tube from left to right: dorsal, right, ventral and left views. Abbreviations: C = crop, EVS = enteric valve seating, MS = mesenteron, MT = mesenteric tongue, P1—P5 = proctodeal segments. Echinotermes biriba, a new genus and species of soldierless termite from the Colombian... 25 Figure 2. Worker enteric valve of Echinotermes biriba sp. n. A Spliced mount B whole mount, showing in situ position of armature. Note the filamentous bacteria attached to the spines. The trilobed seating anterior to the spines (removed in this preparation) is full of bacteria and devoid of food particles, referred by Noirot (2001) as the “bacterial pouch”. 26 Daniel Castro et al. / ZooKeys 748: 21-30 (2018) armature is spherical while in Humutermes it is rather flat. Humutermes species are smaller than Echinotermes. The enteric valve of Grigiotermes is composed of six uniform pectinate plates, while in Patawatermes the uniform plates are hemispherical. Etymology. From the Latin Echino, meaning spiny, describing the EV armature. Echinotermes biriba Castro & Scheftrahn, sp. n. http://zoobank.org/IF9BC8F4-57E9-4608-BB48-FBE9E481940B Holotype. Worker from colony CATAC 2736. Type-locality. COLOMBIA: Caqueta, Belén de los Andaquies (1.60794, -75.88683). Paratypes. PERU: Pasco, Oxapampa, Chatarra forest, (-10.51303, -75.07276), 24/05/2014, 556 m, 14 workers (UF no. PU 144). Additional material: COLOM- BIA: Caqueta, Belén de los Andaquies, Camino Andaqui (1.60794, -75.88683), 31/01/2017, 625 m, 10 workers (CATAC 2736). Description of worker. (Fig. 1, Table 1) EV armature consists of six prominent spheroids each covered with robust spiny armature; three larger (ca. 30-35 spines) and three smaller (15—20 spines) alternate inside the EV seating. Enteric valve with six unsclerotized cushions some four times longer than wide, each composed of approx. 10-20 ovoid scales. Diagnosis. Unique armature of EV composed of alternating larger and smaller spheroids covered with robust spines. Remarks. See genus remarks above. Ecology and distribution. In Colombia, £. biriba foragers were collected in the same soil sample (0-10 cm depth) with Longustitermes manni. Gut contents confirm that £. biriba feeds on soil organic matter. This species is only known from the Cha- tarra forest in the southern Peruvian Amazon, and in a mature secondary forest in the northern Colombian Amazon (Fig. 4). Molecular analysis. The gene tree recovered the Neotropical Apicotermitinae (NA) as monophyletic, however, the position of Echinotermes biriba inside this clade could not be established with this single gene. The low posterior probability of almost Table |. Measurements (mm) of ten workers from two colonies of Echinotermes biriba sp. n. PU144 CATAC2736 Holotype Range Mean Range Mean Max Head Width 0.74 0.77-0.74 0.75 0.83-0.74 0.78 Pronotum Width 0.49-0.46 0.44—0.55 0.51 Hind Tibia Length 0.53-0.44 0.61—0.55 0:57 Fore Tibia Length 0.44—0.35 0.49-0.43 0.46 Fore Tibia Width 0.13 0.14-0.11 Osi? 0.14—0.11 OQ. Fore Tibia Width: Length Ratio 0.27 0.36- 0.24 0.30 0.23-0.28 0.26 Echinotermes biriba, a new genus and species of soldierless termite from the Colombian... 27 Heterotermes_crinitus_KF430191 Macrotermes_bellicosus_AY127702 a —_ Na sutitermes_octopilis KF430192 noe eS Silvestritermes_minutus_KT215789 : Syntermes_grandis_EU253863 0,73 Termes_hispaniolae_FJ802753 Euhamitermes_hamatus_KY224490 Duplidentitermes_sp_KP026271 1 , Jugositermes_tuberculatus_KY224532 | Trichotermes_ducis_KY224506 0,79 Astalotermes_murcus_KY224676 0 os 138 Acholotermes_chirotus_KY224688 : Ateuchotermes_retifaciens_KY224497 —__¥_tH+—1_—————. Alyscotermes_kilimandjaricus_KY224395 1 | Rubeotermes_jheringi_KF430151 ' Rubeotermes_jheringi_KT215778 1 [ Anoplotermes_parvus_HQ398189 Anoplotermes_parvus_HQ398187 ; Amplucrutermes_inflatus_KT215783 oS Patawatermes_nigripunctatus_KT215786 5 eos alls 0,48 0,76 0,41 0,33 0,47 Patawatermes_nigripunctatus_KY683186 Patawatermes_turricola_KY683189 — Patawatermes_turricola_KY683191 0.99 Humutermes_krishnai_KT215787 ; , 0,86 Anoplotermes_janus_KY683193 4,3 '— Anoplotermes_janus_KY683187 '— Anoplotermes_janus_HQ398188 Echinotermes_biriba_UF_PU144 1 Anoplotermes_banksi_KT215785 Anoplotermes_banksi_HQ398185 5 eS Grigiotermes_hageni_KT215781 Grigiotermes_hageni_KY683200 Grigiotermes_hageni_KY683196 Disjunctitermes_insularis_KY683195 '— Disjunctitermes_insularis_KY683188 Tetimatermes_sp_KY683198 Compositermes_bani_KM538651 Compositermes_vindae_KM538652 Compositermes_vindal_KM538649 Aparatermes_cingulatus_KY683192 Aparatermes_cingulatus_KY683194 Ruptitermes_reconditus_KM538647 Aparatermes_spA_KT215784 Aparatermes_sivestrii_KY683197 Aparatermes_silvesinii_KY683190 Hydrecotermes_kawaii_KT215788 ro Longustitermes_manni_KF 430187 Longustitermes_manni_HQ3981 86 oe Longustitermes_manni_KF430083 0,03 Figure 3. Bayesian gene tree of all described soldierless New World genera using the mitochondrial COI barcode gene showing posterior probabilities. Tree rooted on terminal Heterotermes crinitus. every first branching clades in the NA group should be interpreted as a big polytomy, and the new genus as a branch in this polytomy, just as most of the other NA genera (Fig. 3). Etymology. The species name is due to the resemblance of the EV armature with the Amazonian fruit Rollinia mucosa (Jacq.) Baill. which is known as “biriba” in the region. Discussion Neotropical soldierless termites have been a taxonomic problem to a large extent be- cause enteric valve (EV) morphology was overlooked. Mathews (1977) showed it was possible to differentiate some New World Apicotermitidae using the EV as had already 28 80°0'0"O 1 0°0'O" , aa BS aw ait ‘ ie en ek € Ay Sv ECU, ADO R Guayaquil Fe i *, 10°0'0"S 80°0'0"O ; Cartapene g ze J ? S| We Medellin %, © ag es Cucuta Lei et A pecans Ler iA be Pic ‘, pig Daniel Castro et al. / ZooKeys 748: 21-30 (2018) 70°0'0"O laracaibo a fi eas > Barquisimeta, ae Puls % VENEZUELA % 3 a Sie Ayacucho ic Vents. ULANA ile ae s f | youre ‘ ! * oer a j ameetA MA Z OON Iquitos ¢ i yw or Pana Porto Velho hsm a ha je 6 ae 10 420 630 te 60°0'0"O ; GUY. 10°0'0"S Kilometers MATO q | vi. wee = te oe Sources: cart cHieRe, 0 DeLonne, eae increment P Com., GEBCO-OSES-fAO, NPS, NREAN .GeoBae TEN, © NM a renee ates Japan, METI, Esti China (Hong K 2 penstectitap contrib ae ae the GIS User Community 70°0'0"O Figure 4. Known localities of Echinotermes biriba sp. n. ongi, swisslope. M Maprnylnci 60°0'0"O been done in Africa (Grassé and Noirot 1954, Sands 1972), thus furthering the reclas- sification of the so-called Anoplotermes-group to this day. As with D. insularis (Schef- frahn et al. 2017), E. biriba is described only from the worker caste with the EV as its most robust diagnostic character. The Amazon forest contains the greatest diversity of New World termites (Acker- man et al. 2009, Constantino and Cancello 1992), but currently the data show a low diversity of Apicotermitinae compared to other subfamilies such as Nasutitermitinae, Syntermitinae, and Termitinae (Constantino 1991, de Souza and Brown 1994). As new genera and species of neotropical Apicotermitinae are described, the richness of termites, especially in poorly studied countries such as Colombia and Peru will greatly increase. Echinotermes biriba, a new genus and species of soldierless termite from the Colombian... 29 Acknowledgments We want to thank to the Colciencias, Instituto Amazénico de Investigaciones Cienti- ficas SINCHI and Expedicién BIO project for the financial support in Colombian Amazon. We also thank to Dr. Clara P. Pefia-Venegas for coordinating the soil com- ponent at SINCHI and for her logistic support on field trips. We heartily thank the reviewers for their help in improving the paper. 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