1. Introduction
Syllidae is a highly diverse family of the polychaetes (Annelida, Phyllodocida), with 72 described genera and almost 700 species (San Martín, 2003; Aguado & San Martín, 2009; Aguado et al., in press), and continuously new taxa are being described. They are small marine worms, usually of few mm long, although some species can reach up to 90 mm. Contrariwise to their small size, they are very complex, with a body exhibiting numerous structures, external and internal, some of them difficult to examine properly under light microscope, even using higher magnifications and Nomarsky system of polarized light. Description of most species before around the year 2000 was based only on examinations and drawings made with camera lucida under light microscopes. The use of SEM to study syllids is relatively recent, but produced the discover and descriptions of a number of new, unknown structures, or only incompletely known before, whose physiology and significance open a new field of research. The oldest SEM picture of a syllid is from 1980 in which Heacox showed the head of a
Detailed morphological and reproductive observations under SEM had important relevance to the knowledge of the family Syllidae for two reasons.
The first one is related with the phylogeny of the family; discoverment and descriptions of these structures and details of the reproductive modes provided more morphological features to analyze and, consequently, more robust hypothesis about the relationships among the different genera of the family.
The second one is the great help to differentiate sibling complexes of species; species apparently identical morphologically can be differentiated by minute details, only perceptible under SEM.
Two books about polychaetes also includes excellent SEM pictures of Syllidae (as well as many others of different families of polychaetes): Rouse & Pleijel (2001), which shows 6 photos with details of morphology and reproduction, and Beesley et al. (2000) in which a couple of SEM photos about syllid reproduction are shown.
2. Material and methods
Syllids are generally of small size and hence, the process of preparation is usually complex and sometimes difficult, so, it is desirable to prepare several specimens for examination. At least, one to be examined dorsally and another one ventrally; specimen with exerted pharynx is also strongly recommended to examine and take photos of details of the anterior end of the pharynx and their armature, which are important characters for identification to genus level.
Minute specimens are very easily lost during the process of preparation, so it is important to be extremely careful, especially having a short number of specimens.
Fixation of the specimens is a very important process for taking good and sharp pictures. Specimens of syllids are usually dirty, especially those of the genera with dorsal papillae, which produce a sticky secretion which agglutinate debris. However, details of papillation is an important taxonomic trait for identification of species and therefore, it is necessary to clean up them, using a brush of a single, slender hair.
Techniques could be different depending upon the authors and the type of SEM used. The specimens for taxonomic studies are usually fixed in formalin, examined in pure water and finally stored in 70% ethanol. To be prepared for SEM, the selected specimens experience a series of progressive baths in more concentrated alcohol (80%, 90%, and pure ethanol), then dried on critical point and covered of a coat of gold. Rouse & Pleijel (2001) recommend Osmium tetroxide (OsO4) as preferred fixative, although its high toxicity (see Rouse & Pleijel, 2001, p. 7).
3. Results
3.1. Morphology
3.1.1. Ciliation
Many syllids are provided with numerous cilia, whose arrangement has taxonomic importance, but they are difficult to see under light microscope; sometimes, it is possible to observe the presence of tufts of cilia in some appropriate areas, but a detailed description of the arrangement of these cilia is almost impossible or extremely difficult. However, well prepared specimens show, under SEM, a system of transversal rows of cilia, which can be single (fig. 1A) or double (fig. 1B), sometimes some anterior segments with single and from one segment backwards being double (fig. 1B), sometimes only tufts of cilia on some areas; in some species only the peristomium is dorsally provided with a single band of cilia and remaining segments lack them. These details were not included in the descriptions until recently, and they are certainly useful for segregation of species and even similar genera. Descriptions arrangement of ciliary bands, based on SEM examinations are in San Martín (2003) for the species
3.1.2. Nuchal organs
Nuchal organs are only present in polychaetes and are thought to be a synapomorphy for the group (Rouse & Fauchald, 1997; Rouse & Pleijel, 2001), although they show different chemoreceptor structures and are different in shape among the families. The most typical shape appears as two semicircular, densely ciliated pits between prostomium and peristomium; these are the kind of nuchal organs most common in the Syllidae, in fact most genera have the typical nuchal organs (fig. 2A), sometimes extending laterally to prostomium, forming two semicircular ciliated areas (fig. 2B) as in the genera
In the species
3.1.3. Papillation
Some genera of the family Syllidae, as
3.1.4. Pharyngeal armature
The pharyngeal armature in syllids is an important diagnostic character at different taxonomic levels; for this reason, a careful examination of the pharyngeal armature is always necessary for identification. SEM photos help considerably in these observations, and also contributed greatly to the discover of some overlooked details, as ciliation and presence of pores on the pharyngeal papillae, coats of cilia on the pharyngeal opening, secondary crown of papillae, total absence of papillae, etc. The pharynx of syllids can be unarmed or provided with a single middorsal tooth (fig. 4A), usually surrounded by a crown of papillae, and sometimes with a complete or incomplete crown of teeth (trepan) (fig. 4B) sometimes there is a trepan but not a pharyngeal middorsal tooth; teeth of trepan are usually of the same size, but sometimes they are of different sizes (figs. 4C, D). Detailed observations under SEM of the pharyngeal armature, papillae and ciliation are in numerous papers: Capa et al. (2001); Tovar-Hernández et al. (2002); Nogueira & San Martín (2002); Martin et al. (2002; 2003; 2009); San Martín (2003; 2005); San Martín & Hutchings (2006); Aguado & San Martín (2007,2008); Nogueira & Fukuda (2008); San Martín et al. (2008a, b; 2010); Lattig & Martín (2009; in press a, b); Lattig et al. (2007; 2010 a, b); Ramos et al. (2010); Olivier et al. (2011); Salcedo-Oropeza et al. (2011).
3.1.5. Chaetae
Chaetae have a great value for taxonomic identifications; small details of chaetae can be crucial to assign one specimen to one species. Therefore, a careful examination of chaetae is decisive. They are usually the most photographied structures of syllids; together with the pharyngeal armature. The chaetae are the unique hard structures in syllids, so they are relatively easy to be examined and photographied by SEM; however, they are not always clean enough. Additionaly, length and shape can vary from anterior to posterior part of body and from dorsal to ventral. In our knowledge, the oldest publications showing chaetae of any syllid are those of Heacox (1980) and Pawlik (1983) cited above. Later, Westheide (1990) showed the chaetae and also a special genital chaeta of a new species (
Images of the same chaeta under light microscope or SEM can be remarkably different, being the latter much more precise and useful for descriptions and phylogenetic inferences. Also study under SEM of special chaetae for reproduction and brooding of eggs produce interesting discoveries (see below).
3.1.6. Glands
Syllids are provided with numerous kinds of glands, whose function is mostly unknown; some of them were known from long time ago but others were discovered and described recently using SEM. Some of the previously known glands were studied under SEM, and some details of their structures were showed after SEM observations. Most of the different kinds of glands in Syllidae are present in the subfamilies Exogoninae Langerhans, 1879 and Syllinae Grube, 1850, especially in interstitial, minute species, but also in some large syllids. Members of the genus
In some genera of the subfamily Exogoninae (
3.1.7. Other structures
Additionally, SEM, has provided the possibility to find some enigmatic structures, such as transversal lines of spines or papillae in some species of
3.2. Biology and reproduction
Some aspects of the biology has been also remarked using SEM, such is the case of specimens of the genus
Many interesting contributions were made on the external gestation of Exogoninae; Küper & Westheide (1997) demonstrated, including SEM photos, that in the genera
Especially interesting were some photos taken to stolons of the Australian species
3.3. Taxonomy
All these observations, discoveries, and more detailed descriptions of characters, thanks to SEM had important consequences, not only descriptions of new structures, opening new topics for future research, but also in the taxonomy and systematics of this complicate family of polychaetes. Detailed descriptions, using SEM, and comparison of different populations of supposed species of large distribution can help to differentiate cryptic species. The genus
3.4. Phylogeny
All these observations, above explained, under SEM on numerous species of syllids made possible the description of a number of new characters, previously unknown, as well as a better understanding of other features already known. This had important consequences in taxonomy, as already explained, and also on the phylogeny of the family. Sharp and detailed SEM photos are extremely useful to illustrate the characters used for phylogenetic analysis, as done by Nygren (1999), Aguado & San Martín (2009), and Aguado et al. (in press). Nygren (1999) used one plate with six SEM photos to illustrate 8 characters, and Aguado & San Martín (2009) 5 plates with a total of 33 SEM photos, in which ciliation of segments, body shape, ornamentation (papillae, tubercles, crests, among others) nuchal organs and details of nuchal lappets, details of peristomium and prostomium, size and shape of antennae and cirri, palps, parapodia, chaetae, several kinds of glands, pharyngeal armature, kinds of reproduction, etc., were profusely illustrated. The increasement on the number of characters and detailed descriptions supported more robust hypotheses about evolutionary relationships of syllids. The information of new morphological traits or new details about well known structures has been extremely useful to provide evidence for synapomorphies of some clades. For instance, the glands in anterior ventral cirri highly contributed to support a clade with the genera
4. Perspectives and conclusions
The use of SEM in the study of syllids has been proved of high usefulness, showing characters not previously described and for giving appropriate descriptions of other already known. However, since it is also possible the discoverment of new taxa in wide unprospected areas of the world, and a detailed study under SEM still lacks for numerous syllid genera, new morphological structures may still be undescribed. For instance, San Martín et al. (2007) described a new genus and species from Australia (
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