Org Divers Evol (2016) 16:105–119 DOI 10.1007/s13127-015-0257-z ORIGINAL ARTICLE Chaetal type diversity increases during evolution of Eunicida (Annelida) Ekin Tilic1 & Thomas Bartolomaeus1 & Greg W. Rouse2 Received: 21 August 2015 /Accepted: 30 November 2015 /Published online: 15 December 2015 # Gesellschaft für Biologische Systematik 2015 Abstract Annelid chaetae are a superior diagnostic character Keywords Chaetae . Molecular phylogeny . Eunicida . on species and supraspecific levels, because of their structural Systematics variety and taxon specificity. A certain chaetal type, once evolved, must be passed on to descendants, to become char- acteristic for supraspecific taxa. Therefore, one would expect Introduction that chaetal diversity increases within a monophyletic group and that additional chaetae types largely result from transfor- Chaetae in annelids have attracted the interest of scientist for a mation of plesiomorphic chaetae. In order to test these hypoth- very long time, making them one of the most studied, if not the eses and to explain potential losses of diversity, we take up a most studied structures of annelids. This is partly due to the systematic approach in this paper and investigate chaetation in significance of chaetal features when identifying annelids, Eunicida. As a backbone for our analysis, we used a three- since chaetal structure and arrangement are highly constant gene (COI, 16S, 18S) molecular phylogeny of the studied in species and supraspecific taxa. Aside from being a valuable eunicidan species. This phylogeny largely corresponds to pre- source for taxonomists, chaetae have also been the focus of vious assessments of the phylogeny of Eunicida. Presence or many studies in functional ecology (Merz and Edwards 1998; absence of chaetal types was coded for each species included Merz and Woodin 2000; Merz 2015; Pernet 2000; Woodin into the molecular analysis and transformations for these char- and Merz 1987). Furthermore, the cellular mechanisms behind acters were then estimated using the mK1 likelihood model. the chaetal formation process described by Bouligand (1967) Our results show that chaetal type diversity does indeed in- and O’Clair and Cloney (1974) have been an intriguing field crease within eunicids and provide possible explanations for of study. Chaetae are extracellular, chitinous structures formed the homology, convergence, and loss of chaetal types in within an ectodermal pouch, the so-called chaetal follicle. The eunicidan subtaxa. basalmost cell within this follicle is the chaetoblast (Bartolomaeus 1998; Bouligand 1967;Hausen2005; Schroeder 1984;SpechtandWestheide1988). This cell pos- sesses apical microvilli which release N-acetylglucosamine into the intermicrovillar extracellular space where it subse- Electronic supplementary material The online version of this article (doi:10.1007/s13127-015-0257-z) contains supplementary material, quently polymerizes to elongate the chaeta. Pattern, diameter, which is available to authorized users. and number of microvilli continuously change during chaetogenesis, so that the structure of the chaeta reflects these * Ekin Tilic temporal changes and are nothing but cell surface dynamics [email protected] frozen in time (O’Clair and Cloney 1974). This dynamic formation modality presumably allowed 1 Institute of Evolutionary Biology and Animal Ecology, University of the high diversity of chaetal types to evolve. On the Bonn, An der Immenburg 1, 53121 Bonn, Germany other hand, since chaetal arrangement and structure are 2 Scripps Institution of Oceanography, UCSD, 9500 Gilman Drive, La highly taxon-specific, chaetogenesis must be under strict Jolla, CA 92093, USA regulation. Structure, orientation and number of 106 E. Tilic et al. microvilli of the chaetoblast, and modulation of these Material and methods factors determine chaetal structure, and their regulation must be conservative enough that, once evolved, a cer- Animals tain chaetal type can be passed on to descendants, be- coming characteristic for supraspecific taxa. We therefore Specimens of Lumbrineris tetraura (Schmarda, 1861), assume that once evolved strong functional constraints Lumbrineris (Scoletoma) fragilis (O.F. Müller, 1776), must be responsible for fixing certain chaetal types with- Eunice (Leodice) torquata (Quatrefages, 1866), Marphysa in species and supraspecific communities. There is some belli (Audouin & Milne-Edwards, 1833) and Arabella iricolor experimental evidence that such functional constraints (Montagu, 1804) were collected in the intertidal zone during actually exist (Merz 2015). If this were true, one would field trips to Concarneau, France (Brittany). Ophryotrocha sp. expect that certain chaetal types are positively selected n. was collected from aquaria of the Scripps Institution of and maintained in a group of descendants and that alter- Oceanography. Diopatra neapolitana Delle Chiaje, 1841 ation in functional constraints lead to a partial or com- was extracted from tubes collected in Zostera beds during plete transformation of chaetae within this group. low tide in the bay of Arcachon close to Le Petit Piquey Chaetae within a monophyletic annelid taxon should (France) in 1995. show (1) that during evolution, chaetal diversity in- creases within that group and (2) that additional chaetae Scanning electron microscopy types largely result from transformation of chaetae that are present in basally branching taxa. Since there is also Specimens used for scanning electron microscopy (SEM) evidence that certain species secondarily show a rather were fixed in Bouin’s fluid. These were dehydrated in an uniform and simple chaetation (see for instance Aguado alcohol series and were kept in a 5 % phosphotungstic acid et al. (2013) for Chryopetalidae), one also has to ask solution for an hour to increase the heavy metal content in the how chaetal diversity gets lost. tissue. They were critically point-dried with CO in a critical In order to test both hypotheses and to explain potential 2 point dryer (Balzers) and sputtered with gold (Balzers Sputter losses of diversity, we take up a systematic approach in Coater). The specimens were examined in Novoscan and this paper to investigate the distribution of different chae- Leitz AMR 1000 scanning electron microscopes. During de- tal types in Eunicida. Eunicida (sensu Fauchald 1977)isa hydration, the animals were sonicated to remove debris and species-rich taxon (over 900 nominal species in 100 gen- sand particles from the chaetae. era (Rouse and Pleijel 2001)) of annelids and its mono- The Arabella iricolor specimen was not treated with phos- phyly has been established by molecular (Struck et al. photungstic acid and was dehydrated using HMDS, according 2006) and morphological analyses (Rouse and Fauchald to the method described by Nation (1983). This specimen was 1997). The characteristic autapomorphy for Eunicida is analyzed using a Philips XL30 ESEM. the cuticular, prominent jaw apparatus composed of mul- tiple elements (Purschke 1987). Eunicida are presently classified into five major subtaxa: Lumbrineridae, Confocal laser scanning microscopy Oenonidae, Dorvilleidae, Onuphidae, and Eunicidae, as well as two minor groups, the obscure Hartmaniellidae The specimens used for confocal laser scanning microscopy and the symbiotic Histriobdellidae. (CLSM) were fixed in 4 % paraformaldehyde. In most of the Eunicida show a variety of different chaetal types that studied species, chaetigers were dissected to separate single range from simple capillaries to more complex hooded parapodia or single segments. The specimens were perme- hooked or compound chaetae. We therefore investigated the abilized in four 5-min changes of phosphate-buffered saline chaetae of a range of Eunicida and conducted a thorough (PBS) with 0.1 % Triton X-100 (Fisher Scientific). The survey of chaetal types described in the literature. As a back- parapodia were then stained overnight in 4 °C with TRITC bone for our analysis we used a three-gene (mitochondrial phalloidin at a dilution of 1:100. After staining, parapodia COI, 16S rDNA and nuclear 18S rDNA) molecular phyloge- were rinsed in three quick changes and subsequently in two ny of Eunicida that includes all species we used to analyze 10-min changes of PBS with 0.1 % Triton and one 10-min chaetal diversity and arguably covers the diversity of the rinse in PBS without Triton. Larger samples were dehydrated group. We did not include Hartmaniellidae, since no speci- in isopropanol (2 min 70 %, 2 min 85 %, 2 min 95 %, 2 min mens of were available for analysis or sequencing. The posi- 100 %, 2 min 100 %), cleared in three 15-min changes of tion of Histriobdellidae cannot be resolved with certainty due Murray Clear, and mounted in hollow-ground slides with to the low amount of sequence data available. Histriobdellids Murray Clear. All coverslips were sealed with nail polish. A do not bear any chaetae and the loss of chaetation in this taxon Leica TCS SPE laser scanning confocal microscope was used is discussed. for the analysis. Chaetal type diversity increases during evolution of Eunicida 107 Light microscopy Eunicidae. The maximum likelihood tree (Fig. 4), largely matched the maximum parsimony analysis in topology and Chaetae of selected species were isolated by using a 5 % support, except for Onuphidae, which it showed to be mono- NaOH solution. After the tissue was completely dissolved, phyletic. Lumbrineridae was the sister group to a well- chaetae were rinsed and studied under an Olympus micro-