Internal ornamentation of the first proctodeal segment of the digestive tube of Syntermitinae ( Isoptera , Termitidae )

The ornaments inside the first proctodeal segment of the digestive tube in thirty-three species of the termite subfamily Syntermitinae are described and illustrated, encompassing all genera of the subfamily. The occurrence, type and pattern of the ornamentation reveal a wide morphological diversity. A first proposal for classification and nomenclature of these structures and coverage patterns is included, as well as a discussion of possible functions.


Introduction
The termite gut is specialized for the digestion of lignocellulosic resources.Unlike other insects, the hindgut is compartmentalized, and five segments can be recognized: ileum, or first proctodeal segment (P1); enteric valve, or second proctodeal segment (P2); paunch, or third proctodeal segment (P3); colon, or fourth proctodeal segment (P4); and rectum, or fifth proctodeal segment (P5) (Noirot 1995).During the evolution of termites, many groups evolved more anatomically and physiologically complex adaptations that allowed them to diversify their diet.For example, members of the subfamily Cubitermitinae, a well-characterized soil-feeder group, have a specialized diverticulum connected to the P3, and a complex physiology that regulates the pH and the oxygen supply in the gut compartments (Brune 2014).The digestive process, mediated by symbionts, occurs mainly in the P3, but the entire gut is important for digestion.
The gut characters are important for termite taxonomy.One of the first comprehensive studies employing gut anatomy was that of Sands (1972), who reorganized a complex of soldierless termite species and described 16 new genera, based mainly on these characters; additionally, Sands revamped the entire classification of Termitidae.Although Sands never employed the P1 ornaments, his work is a good example of how the morphology of the gut can be taxonomically informative for Isoptera.Although the internal ornaments have been widely used in termite systematics, the major focus has been on P2, and published information about other segments is sparse.Kovoor (1969) contributed the first comparative study of the anatomy of the digestive tube of termites, including species of Syntermitinae (at that time considered as part of Nasutitermitinae).She described the gut of Cornitermes cumulans, Labiotermes pelliceus, Procornitermes triacifer, Rhynchotermes nasutissimus, Syntermes dirus and an unspecified species of "Armitermes" (probably Silvestritermes euamignathus), emphasizing important morphological differences between the "mandibulate nasutes" and "true nasutes", which today are assigned to the Syntermitinae and Nasutitermitinae, respectively.Kovoor (1969) mentioned the existence of ornamentation inside the (P1) in Syntermitinae, describing and illustrating this ornamentation for Syntermes dirus (only by a small indication, without details) and describing a slightly different arrangement of spines for other species of the Syntermes-group.However, she did not discuss the possible significance of the differences or make a detailed microscopic examination of the ornamentation.
The papers of Noirot (1995Noirot ( , 2001) ) systematized the knowledge of termite gut anatomy, establishing a nomenclature and comparing a wide range of species from all families and subfamilies of Isoptera, and also the wood roach Cryptocercus, the sister-group of termites (Krishna et al. 2013).Noirot (1995) described the first proctodeal segment in lower termites.In these groups the P1 is very short and has six internal cushions, with tri-radial symmetry and ornamented with small spines, similar to Cryptocercus.Noirot (2001) indicated that the ornamentation, although reduced, persists in the subfamily Macrotermitinae, but for the other groups of Termitidae it is not clear if there is a pattern.
Except for the study of Kovoor (1969), the P1 internal ornaments are neglected in termite literature, particularly for species of Termitidae.Even in the most comprehensive termite treatises that include information about the gut anatomy (Grassé 1982;Krishna et al. 2013;Noirot 1995Noirot , 2001)), the existence of these ornaments is only mentioned, and there is no specific description, categorization or hypothesis respecting their function.As mentioned above, Noirot (1995Noirot ( , 2001) ) described a generalized pattern for the families of lower termites, but except for few notes about Macrotermitinae, almost nothing is mentioned for Termitidae, which comprise two-thirds of Isoptera species.Besides, Termitidae have a much larger food repertoire compared with the lower termites, that feed strictly wood (Brune 2014).
Herein we describe and compare the internal ornamentation of the first proctodeal segment for a comprehensive set of Syntermitinae species, and propose, for the first time, a comparative nomenclature for these structures and coverage patterns.There is wide variation within the subfamily, and insights about the taxonomic value, classification by feeding habits, and function in termite digestion are discussed.

Materials and methods
The specimens studied are deposited in the Museu de Zoologia da Universidade de São Paulo (MZUSP), São Paulo, Brazil.We dissected individuals of 36 species of Syntermitinae, covering all genera (Table 1).These represented nearly one-third of the 101 described taxa of Syntermitinae, and were chosen by a previous examination of all subfamily species to include the most representative.
The arrangement of the ornamentation is represented by schematic drawings.Since in the Syntermitinae the P1 is inflated and globose, it is impossible to photograph or illustrate the complete coverage exactly as it exists in situ and on the actual scale.The plan of the dissections is explained in Fig. 1.
The structures were removed, mounted in glycerin, and photographed under an optical microscope (Zeiss Microscope Axionskop 2 coupled to a computer).The terminology adopted for the digestive tube follows Noirot (2001).

Results
The analysis of the P1 ornamentation revealed a wide diversity of structures and patterns .The structures can be categorized by form and relative arrangement, and the ornamentation by the types of spines and their abundance.As a specific nomenclature for these structures does not exist, we propose some terms that are discussed below.
Robust spines are relatively short (around 10 µm), with a simple conical shape.They are sparsely distributed (Figs 16b,25b).Stout spines are similar to robust spines, but larger and sometimes slightly curved; they may be arranged longitudinally (Figs 11c,16c) or regularly alternating (Fig. 12d).
The central ridges, present in some species of Labiotermes (Figs 13c,14c,15c), are single structures, composed of spines with their bases fused, arranged in a helical pattern.These ridges are probably homologous to the longitudinal arrangement of stout spines in Ibitermes curupira and Mapinguaritermes (Figs 11c, 16c), since their placements are equivalent inside P1.
Small spines in rows ( Figs 4c, 21c, 23c, 23f) are tiny and have a typical transverse arrangement in units of 2-6 spines.

Patterns of ornamentation
The coverage and abundance of spines can be categorized in four types of patterns.
Cyrilliotermes angulariceps (Fig. 6): The coverage is composed only by small aciculiform spines, one group located just after the mesenteric tongue (Fig. 6b), and three ovate areas are present at the distal region (Fig. 6c).Embiratermes brevinasus: identical to E. ignotus (see ahead).Embiratermes festivellus (Fig. 10): A triangular area of aciculiform spines is located just after the mesenteric  tongue (Fig. 10b), and three oval areas, covered with small aciculiform spines, present at the distal region.Embiratermes ignotus (Fig. 8): The only ornament is a single transversal row of trifurcate spines, placed laterally to the mesenteric tongue (Fig. 8b).Embiratermes silvestrii (Fig. 9): An oval area of aciculiform spines is located laterally to the mesenteric tongue (Fig. 9b), and three branches of small spines, one long and two shorts, are present at the distal region (Fig. 9c).Ibitermes tellustris: very similar to E. silvestrii (see above).
Macuxitermes triceratops: (Fig. 17): An elongated area of aciculiform spines is placed close and laterally to the mesenteric tongue (Fig. 17c), the distal portion is broadly covered by sparse small spines (Fig. 17b).Noirotitermes noiroti (Fig. 18): A group of aciculiform spines is located laterally to the mesenteric tongue (Fig. 18c), and at the distal region three long branches of small spines are present (Fig. 18b).Paracurvitermes manni (Fig. 20): A transversal row of aciculiform spines, placed laterally to the mesenteric tongue is located laterally to the mesenteric tongue  (Fig. 20c), and three branches of small spines, one long and two shorts, are present at the distal region (Fig. 20b).Silvestritermes euamignathus (Fig. 22): A triangular area of aciculiform spines is located just after the mesenteric tongue (Fig. 22c), the aciculiform spines form a singular palisade around the mesenteric tongue (Fig. 22b).Three branches of small spines, one long and two very short, are present at the distal region.row of 20-25 stout spines (Fig. 11c), and few robust spines spaced distributed around the stout spines.Labiotermes emersoni (Fig. 12): At the proximal region, between the mesenteric tongues, three longitudinal rows of robust spines (Fig. 12c) with the central ridge at the apex of the middle row (Fig. 12d), distal region covered with spaced small bristles (Fig. 12b).Labiotermes labralis (Fig. 13): The internal surface is all covered with spaced robust spines (Fig. 13b), with a small central ridge (Fig. 13a).
Labiotermes leptothrix (Fig. 14): The central ridge is "S" shaped, well sclerotized with the fused spines (Fig. 14a), distal region covered with spaced robust spines.Labiotermes orthocephalus (Fig. 15): The central ridge is composed by 10-15 slightly fused spines (Fig. 15c); distal region is covered with spaced robust spines (Fig. 15b).Mapinguaritermes peruanus (Fig. 16): The ornaments are concentrated laterally to the mesenteric tongue, a longitudinal row of 15-20 stout spines (Fig. 16c), and robust spines spaced distributed around.2c).Embiratermes heterotypus (Fig. 7): All surfaces are covered with a layer of aciculiform spines (Fig. 7b).Broad coverage: characterized by a dense coverage of spines (Figs 4a, 21a, 23a, 24a), mainly aciculiform, and small spines in rows, with recurrent sclerotized spines, the following species can be included in this category: Cahuallitermes intermedius: very similar to C. cumulans (see ahead).Cornitermes cumulans (Fig. 4): The surface is covered by different types of spines, laterally to the large mesenteric tongue an elongated area of aciculiform spines (Fig. 4b), the distal region and the edge of the mesenteric tongues are covered with small spines in rows (Fig. 4c), the remain surfaces are covered with spaced robust spines.Cornitermes silvestrii, Procornitermes araujoi, Procornitermes triacifer: very similar to C. cumulans (see above).Rhynchotermes nasutissimus: very similar to R. perarmatus (see ahead).Rhynchotermes perarmatus (Fig. 21): laterally to the mesenteric tongue there is an elongated area covered with strongly sclerotized aciculiform spines (Fig. 21b), the distal region is covered with small spines in rows (Fig. 21c).Syntermes molestus (Fig.  23f) and the remain surfaces are covered with spaced robust spines.Syntermes spinosus (Fig. 24): laterally to the mesenteric tongue, an elongated area covered with robust spines (Fig. 24b), with a small area with strongly sclerotized aciculiform spines at the middle (Fig. 24c), the edges of the mesenteric tongues are covered with small spines in rows.
The species A. armiger, A. bidentatus and U. teevani (Fig. 25) does not fit clearly in the previous proposed  patterns, at the proximal region there is a irregular area covered with robust spines (Figs 25b, 25c), but without the longitudinal row of stout spines or the central ridge, and the distal region is covered with a single longitudinal and large row of spaced small spines, that can not be clearly recognize as equivalent to the broad coverage or the three branches of the proximal aciculiform pattern.
In a few of the species studied, the ornament coverage is reduced to short areas with small, sclerotized, aciculiform spines, i.e. in C. acignathus, C. bolivianus (Fig. 3) and P. lespesii (Fig. 19).
The ornamentation pattern in Syntermitinae is bilaterally symmetrical or asymmetrical (in a few species).The majority of genera of Syntermitinae have a consistent pattern of ornamentation among their species; the exceptions are Ibitermes, Embiratermes and Procornitermes, particularly Embiratermes.This indicates that these genera are not monophyletic, in agreement with observations of other gut anatomical characters (Cancello and Rocha 2013, for Procornitermes; personal observation for Embiratermes and Ibitermes, manuscript in preparation).
Although these structures may have potential phylogenetic information, their relevance as characters and the congruence with the gut anatomy need to be checked in most comprehensive studies.A preliminary phylogeny of Syntermitinae was proposed in Rocha et al. 2012, but the main objective in the study is just formulate a working hypothesis to test the monophyly of Armitermes, a more specific study is necessary to make phylogenetic inferences about the evolution of the ornaments.

Feeding habit inferences
In studies of termite faunas, the species are commonly classified by diet.The categories are defined by a humification gradient, which ranges from whole plant matter (such as wood and dead leaves) to decomposed cellulosic compounds dispersed in the soil (see Donovan et al. 2001 for a more detailed description).Although the classification by feeding groups is widely used, more recent studies have indicated that there is a continuum among termite feeding preferences (Bourguignon et al. 2010).
No specific proposal has been made for a classification of the feeding habits of all Syntermitinae species.Based on field notes accompanying specimens deposited in the MZUSP, and inferences from worker mandibles and gut morphology, we made a compilation and a classification for their diet, and compared feeding habits and P1 patterns of ornamentation among the species examined (Table 2).
The pattern of ornamentation in Syntermitinae appears to be generally related to the feeding habit, and reflects the humification gradient.Species that feed lower  in the humification gradient (litter-feeders, grass-feeders and wood-feeders) mainly display a mosaic pattern, with a wide and heterogeneous coverage of small spines (for example Syntermes, Cornitermes, Rhynchotermes).Species that feed on humidified resources (intermediateand soil-feeders) have a more centralized pattern, with structures concentrated only in the proximal region of P1 (for example Silvestritermes, Curvitermes, Mapin-guaritermes, Labiotermes) (see Table 2).This inference may be useful as an additional character to determine the species' diet, combined with other characteristics of the gut.However, some of the species examined in this study are exceptions; for instance, P. lespesii and C. bolivianus are known as, respectively, wood-feeder and litter-feeder species, although their P1 ornamentation is much reduced.

Function in termite digestion
As the P1 ornaments have never been described in detail for Termitidae species, we can only speculate as to their function.Investigating their specific function is outside the scope of this paper, but we can make some suggestions based on morphological inference and analogies with the functions of P2 suggested by Donovan et al. (2000).
These structures may function as an abrasive surface for the food mass that enters from the midgut.This hypothesis is supported by the location of the ornaments, only in the proximal part of the convex side of the mesenteric arch (see Fig. 1).However, in some cases the ridges may be irrelevant for this function, for example the isolated central ridges in Labiotermes (Figs 12-15) measure around 50-100 µm and the P1 diameter varies between 1-2 mm, and it is difficult to explain how a single structure of this size can act on the food mass.
Another possible explanation is microbial inoculation.This hypothesis is supported by the usual presence of what appears to be a bacterial film covering the spines, and some points regarding this possibility need to be considered.The pH and oxygenation conditions inside the first proctodeal segment may be very differ-  ent from the third proctodeal segment, which implies that the bacterial flora will also necessarily be different.Bignell and Eggleton (1995) studied the pH conditions inside each segment of the digestive tract in various termite species, and found that P1 may be more alkaline than P3.Schmitt-Wagner et al. (2003) demonstrated for two Cubitermes species (African soil-feeder termites) that inside each hindgut compartment (P1-P5) the symbiont assemblage is composed of distinct types and proportions of microbial groups; this reinforces a microbial-inoculation hypothesis.The mean size of the ornamentation is approximately 20 µm, suggesting an association of the bacteria of the micro-oxic region, the peripheral region of the lumen, of approximately 100 µm, with oxygen availability (Brune and Friedrich 2000).To confirm this, more-detailed studies are needed to bolster this hypothesis.
In other groups of Termitidae, with the same range of feeding habits, ornamentation appears to be absent.For example, in Nasutitermitinae the species have a variety of feeding habits that cover the entire humification gradient, but we never find ornaments in their first proctodeal segment (personal observation).This suggests that in the course of termite evolution, many different strategies have emerged in response to the problems of digesting similar types of food.

Conclusion
Additional information is needed in order to determine the functions and importance of these structures in termite digestion, although the variability among the species clearly shows the taxonomic value of the structure.This character may have taxonomic value in other termite groups, especially for Termitinae and Apicotermitinae, although further studies are needed to confirm this possibility.

Figure 1 .
Figure 1.First proctodeal segment (P1), diagrammatic illustrations showing the line of incision most often used in this study.(a) Silvestritermes euamignathus, mesenteric tongue and ornamentation situated on convex side of mesenteric arch; (b) Rhynchotermes nasutissimus, ornamentation situated on convex side of mesenteric arch and mesenteric tongue twisted.

Figure 4 .
Figure 4. Cornitermes cumulans.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) Aciculiform spines, situated between the mesenteric tongues; (c) Detail of short spines in rows, present around the mesenteric tongues and next to P2.

Figure 5 .
Figure 5. Curvitermes odontognathus.(a) Schematic drawing of P1 showing arrangement of spines; (b) Photomicrograph of set of aciculiform spines, positioned just after the mesenteric tongue.
Figure 11.Ibitermes curupira.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) General view of longitudinal row of stout spines, with some robust spines nearby (middle of P1 wall slightly damaged); (c) Detail of stout spines.

Figure 12 .
Figure 12.Labiotermes emersoni.(a) Schematic drawing of P1 showing arrangement of spines.(b-d) Photomicrographs: (b) Detail of spines in the distal region; (c) General view of the three branches of spines between the mesenteric tongues; (d) Detail of middle branch of spines.

Figure 13 .
Figure 13.Labiotermes labralis.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) Detail of the coverage of small robust spines between the mesenteric tongues; (c) Detail of central ridge structure.

Figure 15 .
Figure 15.Labiotermes orthocephalus.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) Detail of the sparse short spines in the distal portion; (c) Detail of central ridge structure.

Figure 16 .
Figure 16.Mapinguaritermes peruanus.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) General view of the longitudinal row of stout spines, surround by robust spines; (c) Detail of stout spines.
23): similar to C. cumulans, the surface is covered by different types of spines, laterally to the large mesenteric tongue is an elongated area of strongly sclerotized aciculiform spines (Figs 23b, 23d, 23e), the distal region and the edge of the mes-enteric tongues are covered with small spines in rows (Figs 23c,

Figure 17 .
Figure 17.Macuxitermes triceratops.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) General view of the short spines in the distal region; (c) Set of aciculiform spines in detail.

Figure 19 .
Figure 19.Procornitermes lespesii.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) General view showing three proximal sets of small spines; (c) Small spines with sclerotized base in detail.

Figure 20 .
Figure 20.Paracurvitermes manni.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) Detail of the middle distal row of spines; (c) Transverse row of aciculiform spines in detail.

Figure 21 .
Figure 21.Rhynchotermes perarmatus.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) Detail of spines with strongly sclerotized base next to mesenteric tongue; (c) Detail of rows of small spines in the distal region.

Figure 23 .
Figure 23.Syntermes molestus.(a) Schematic drawing of P1 showing arrangement of spines.(b-f) Photomicrographs: (b) General view of the region between the mesenteric tongues; (c) Detail of small spines in rows adjacent to the mesenteric tongue; (d) Detail of spines with strongly sclerotized base between the mesenteric tongues; (e) Detail of strongly sclerotized spines in the middle of the region between the mesenteric tongues; (f) Detail of the small spines in rows in the distal region.

Figure 24 .
Figure 24.Syntermes spinosus.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) General view of sclerotized spines between the mesenteric tongues; (c) Detail of the strongly sclerotized spines with their bases merged.

Figure 25 .
Figure 25.Uncitermes teevani.(a) Schematic drawing of P1 showing arrangement of spines.(b, c) Photomicrographs: (b) General view of the robust spines covering the proximal portion; (c) Detail of robust spines.

Table 1 .
Genera and species studied.

Table 2 .
Association between patterns of ornaments for the species studied, and their feeding habits.