Morphometry and taxonomynext section
Molecular analyses currently tend to displace morphometry in studies trying to discriminate within closely related morphotypes for taxonomic purposes (Blaxter, 2004; Hebert and Gregory, 2005; Godfray, 2007). However, most taxonomists agree that combining the different approaches, applying the so-called Integrative Taxonomy, is the most effective strategy to build a stable and robust taxonomy (Will et al., 2005; Padial et al., 2010). Hence, the importance of morphology must not be forgotten in the genomics era (Giribert, 2015). Moreover, it is not always possible to obtain adequate material for genetic studies because the used preservation methods are destructive, while there is no fresh material available. In such cases morphometry may be the only approach allowing to identify distinguishing characters within species complexes.
Among polychaetes, many taxonomically robust morphological characters may be defined by measurements and/or proportions, some of them being size-dependent (Ben-Eliahu, 1987; Fauchald, 1991; Sigvaldadóttir and Mackie, 1993). This approach has been used independently of molecular analyses to successfully resolve the taxonomy of several sibling species complexes, often leading to the description of new species (Orrhage and Sundberg, 1990; Fauchald, 1991; Blake, 2000; Koh and Bhaud, 2003; Koh et al., 2003; Martin et al., 2003, 2006, 2009; Ford and Hutchings, 2005; Garraffoni and de Garcia Camargo, 2006; Glasby and Glasby, 2006; Lattig et al., 2007; Hernández-Alcántara and Solís-Weiss, 2014; Coutinho et al., 2015). However, Ford and Hutchings (2005) were the first to consider the use of the statistical dissimilarities derived from the SIMPER routine of the PRIMER software (Clarke and Warwick, 2001; Clarke and Gorley, 2006), based on a matrix of morphometric measurements, as a robust support to distinguish between morphologically close species. Accordingly, they described three new species whose average dissimilarities ranged between 11-19%, later finding morphological evidences supporting the erection of the new species. Our morphometric approach, in turn, clearly discriminates the two populations of “O. humesi” which, despite their close morphologies, showed average dissimilarities ranging from 30 to 36% in the SIMPER, thus almost three times higher than the Australian species of Owenia. A detailed comparison of character variability, particularly those that were responsible for the intra-population similarity and the inter-population dissimilarity, led to a reliable way to solve this question. There were significant differences in size range between the Iberian and the Congolese populations (more restricted in the later, leading us to find less size-correlated parameters in Congolese worms). Our approach to compare the two populations led to evident and consistent differences in the appendage measurements and relative proportions (Tables 6, 8) that are considered as robust enough to formally describe the Iberian specimens as the new species O. okupa sp. nov. In addition, we further validate these differences by means of discriminant analyses, whose respective discriminant functions were able to identify the members of the two species with a reliability of 96 to 100% in all tests.
Host/symbiont size relationships. Positive host/symbiont size relationships may either indicate 1) active size segregation behaviour, as reported both for the symbiotic coral dwelling crab Trapezia bidentata (Forskål, 1775) (Adams et al., 1985) and the fish Gobiodon histrio (Valenciennes, 1837) (Hobbs and Munday, 2004), which seem to be able to migrate from colony to colony to choose one of an appropriate size, or 2) parallel growth of hosts and symbionts, as reported both for the nemertean Malacobdella grossa (Müller, 1776), hosted by Arctica islandica (Linnaeus, 1767) (Sundet and Jobling, 1985) and the pontoniin shrimp Anchistus custos (Forskål, 1775), hosted by the bivalve Pinna bicolor Gmelin, 1791 (Britayev and Fahrutdinov, 1994). In turn, the absence of host/symbiont size correlation could be caused by symbionts that either colonize the hosts during a single phase of their life-history (e.g. juveniles, adults), that are highly mobile (thus, colonizing the available host independently of their size), or growth faster than the host, even if the infestation starts with juveniles colonizing small-sized hosts (Britayev et al., 2007).
Among symbiotic polychaetes, positive host/symbiont size-relationships are not common (Martin and Britayev, 1998) and, when occurring, they may be modified by differential behaviour linked to, for instance, reproductive activities. This seems to be the case of Branchipolynoe seepensis Pettibone, 1986, an obligate symbiont of deep-sea hydrothermal vent mytilids of the genus Bathymodiolus, whose females have a longer life span than males and remain inside the same host along their whole life, while males leave their original hosts for mating and then colonize the available hosts independently of their size. Consequently, positive host/symbiont size correlations occur only for females and for non-mature males (Britayev et al., 2007). In most known cases, however, the relationship between size structure of symbiotic polychaetes and their hosts is unclear (Martin et al., 1991, 1992; Emson et al., 1993; Rozbaczylo and Cañete, 1993; Britayev and Zamyshliak, 1996) so that the life histories of the former have been considered to be independent of that of the latter, with the hosts tending to live longer and commonly hosting successive symbionts (Martin and Britayev, 1998).
The absence of host/symbiont size-relationships previously reported for the intertidal population of O. okupa sp. nov. (Martin et al., 2012) was here confirmed by the absence of significant correlations found along the whole study period. The only exception was April 2012, which we consider as a spurious relationship due to the low number of collected worms (n<10) (Fig. 4; Table 9). However, other months with a similar number of individuals (e.g. n = 8 - 11 in March, October, and August) (Fig. 4; Table 9) showed non-significant relationships. These results support Martin et al. (2012) in that the worms do not grow together with their hosts in their intertidal environment. In turn, O. okupa sp. nov. was reported to infest specimens of S. plana > 20 mm in shell length and to be more frequent in intermediate-sized shells, 26-36 mm long (Martin et al., 2012), which has also been confirmed along the whole period of study. Limiting host sizes have been reported for the starfish Asterias rathbuni (Verrill, 1909) in Vostok Bay (Sea of Japan), whose specimens with disc radii lower than 35 mm were not inhabited by Arctonoe vittata (Grube, 1855), and those with radii up to 90 mm harboured one single symbiont, whereas the largest starfishes could host up to four polychaetes (Britayev et al., 1989). Another example occurs in Chile, where the scale-worm Harmothoe commensalis Rozbaczylo and Cañete, 1993 did not occur in specimens of the clam Gari solida (Gray, 1828) < 60 mm in shell length (Rozbaczylo and Cañete, 1993). The size-limit for S. plana as a host of O. okupa sp. nov., also implied that there were no small juveniles infesting small-sized hosts, which allows us to discard the fast-growing hypothesis for this new species.
On the other hand, the infested specimens of M. pellucida collected in a subtidal environment during January 2013 included a considerable amount of small-sized hosts (Fig. 6). Moreover, there was a significant positive size correlation between hosts and symbionts. This relationship may be caused either by the symbiont selecting the host to be infested according to the appropriateness of its size, or by host/symbiont parallel growth, which cannot be assessed in light of our present data. Despite the low number of collected bivalves, the existing size relationship and the very high infestation rates (Table 9), suggest together that M. pellucida and the subtidal environment could be the preferred host and habitat for O. okupa sp. nov., with the symbiont selecting the most suitable hosts when conditions are optimal.
Prevalence of the infestation. The prevalence of commensal polychaetes is highly variable and has been considered as a species-specific characteristic. However, a commensal species may also show different prevalences, which may vary according to bathymetric, spatial and temporal (i.e. intra- and inter-annual) patterns (Martin and Britayev, 1998). Studies including temporal trends are, however, scarce. With the exception of the impressive fidelity of the year-to-year counts of a regular seasonal trend reported for the infestation prevalence of Ophiocoma echinata (Lamarck, 1816) by Branchiosyllis exilis (Gravier, 1900) in Panamá (Hendler and Meyer, 1982), the only known data on year-to-year variability for a symbiotic polychaete are probably those on Arctonoe vittata (Grube, 1855). In Vostok Bay, this worm infests the starfish Asterias amurensis Lütken, 1871 with a progressive increase in prevalence from 0% in 1975-76 to 8.4% in 1978 and to 79.1% in 1980 (Britayev, 1991). Based on our study period, O. okupa sp. nov. seems to show a seasonal trend in prevalence, with the highest percentages occurring during the coldest seasons, i.e. from late autumn to late winter (Fig. 5; Table 9). The present dataset only covers a single year and therefore inter-annual regularities cannot be inferred.
Theoretically, we may expect an influence of the host’s population structure on that of the symbiont (Martin & Britayev, 1998). A positive relationship between host density and infestation characteristics was reported for A. vittata and its host starfish A. rathbuni in Vostok Bay (Britayev et al., 1989). The highest prevalence, mean intensity and abundance depended upon host’s density, which could be caused by the accumulation of a chemically-mediated host-cue more effectively attracting the settling symbionts. High host densities may also reduce the influence of external factors. For instance, commensals associated with less abundant host populations may experience a more relevant decrease in fitness than those harboured by dense host populations (Martin and Britayev, 1998).
Among the few relationships with known data, high infestation indexes tend to be positively correlated with the availability of large and numerous hosts to be occupied by the symbionts (Martin and Britayev, 1998). In the present case, the overall low prevalence would lead to the expectation of a low number of S. plana > 20 mm long in the study area. Conversely, S. plana was very abundant independently of size (Subida et al.,2011; Drake et al., 2014) and so numerous adequate hosts were available through the whole study period (Fig. 6; Table 9). Therefore, the prevalence seems not to be connected with the host availability. In turn, this low prevalence could be related to the extreme daily changes in temperature and/or the alternating long desiccation/immersion periods that characterize the intertidal environment, as well as with biota disturbance due to bait digging of this zone at Río San Pedro (Carvalho et al., 2013). The high prevalence in the subtidal population of M. pellucida (i.e. higher than 85% in specimens > 20 mm long), seems to confirm this hypothesis, with the permanent immersion in the subtidal environment favouring the presence of the symbiont.
The fact that no specimens of S. plana < 18 mm in shell length were found during the annual cycle, is in agreement with all previous data obtained in punctual samplings (Martin et al. 2012, 2015). The present approach did not clarify this peculiarity of the studied bivalve population. However, the high density of specimens restricted to the narrow intertidal area at Río San Pedro led to postulate the existence of a negative interaction between the established population and the settling larvae, which could be actively ingested by their conspecific adults during their normal filter/suspension feeding activities (i.e. passive cannibalism) (Cargnin-Ferreira, 2005; Santos et al. 2011).
The association of a symbiont with various hosts in the same locality could affect the prevalence, which strongly depends on its level of affinity for the different hosts. Although the symbiotic population of O. okupa sp. nov. at Río San Pedro only infested S. plana, this factor could not be eventually discarded due to the presence of the nearby subtidal population of M. pellucida at the opening of Río San Pedro.
Infestation intensity. The infestation intensity of symbiont polychaetes has been more widely reported than the prevalence. It may range from one to hundreds of symbionts per host, but is clearly dominated by the association of a single symbiont per host either due to the usually low symbiont densities or to the influence of an intraspecific aggressive behaviour (Martin and Britayev, 1998). Like the prevalence, intensity may also oscillate within the same population due to seasonal changes in relative abundances, linked or not to reproduction and recruitment events. A common situation for symbionts with 1:1 regular distributions like O. okupa sp. nov., is that adults may occasionally share the host with one to several juveniles, as previously reported for polychaete species of the genera Acholoe, Adyte or Branchiosyllis, among others (Martin and Britayev, 1998).
In the case of O. okupa sp. nov., Martin et al. (2012, 2015) reported single findings of one male and one female and one male, one female and one small worm, likely a juvenile sharing the same host, while the constant regular distribution of one single symbiont per host was attributed to intraspecific aggressive behaviour. There are several cases of symbiotic polychaetes with male and female couples living together associated with the same host, such as the Mediterranean fish parasite Ichthyotomus sanguinarius Eisig, 1906 (Eisig, 1906; Culurgioni et al., 2006) or the deep-sea hexactinellid sponge symbiont Robertianella synophthalma McIntosh, 1885, reported as Harmothoe hyalonemae in Martin et al. (1992). However, in both cases, most hosts harboured couples, while in O. okupa sp. nov. couples seemed to be the exception.
Little is known about the life cycle of most commensal polychaetes and, when known usually does not differ much from that of their free-living relatives. Thus, it is expected to find planktonic larvae (responsible of dispersal and colonization) and benthic adults (with a somewhat reduced mobility) with the single main difference that the symbiotic mode of life replaces the free-living one during the benthic phase (Martin and Britayev, 1998). Larval settlement may occur on the bottom, being then followed by a juvenile migration towards the respective hosts (Davenport and Boolootian, 1966). However, it seems more likely that chemically mediated cues (either generated by the host or by the own symbiotic adults) driving larval settlement could be the most widespread behaviour among symbiotic polychaetes (parasites included) (Martin and Britayev, 1998).
As it occurs for the free-living species, this basic life-cycle scheme may vary in many ways as a result of the adaptation to a symbiotic mode of life. For instance, it can be simplified by reducing (or even eliminating) the free-living pelagic stage or become more complex by having one or more intermediate hosts, which are occupied when the preferred ones are not available or because they have more room to host several juveniles during growing period (Martin and Britayev, 1998). Herewith, intraspecific competition and aggression may play a major role in the associated relocation processes. In the case of O. okupa sp. nov., the only information known on the juveniles is that they have not been found inside S. plana, neither free-living, which has been confirmed during the studied period.
The highest number of small worms found inside S. plana occurred in late autumn, which could be considered as an indication of new symbiont’s recruitment into the host bivalves (after the population being more actively reproducing during summer). In turn, large adults tended to disappear around mid-winter. This may suggest that the life span of O. okupa sp. nov. may be of one year, with the adults dying after reproducing. However, sampling during successive years would be required to confirm this hypothesis, as well as to assess the regularity of the recruitment events.
The life cycle of O. okupa sp. nov. may be limited by the tidal regime characteristic of its intertidal habitat, while the highly abundant population living in M. pellucida seems to indicate that recruitment may occur mainly in the more favourable subtidal conditions. Thus, we propose a possible scenario in which the symbionts mainly inhabit certain areas of the Bay not submitted to periodical desiccation by tides, living in association with M. pellucida (but we cannot discard other possible unknown hosts), then reaching intertidal areas such as those at Río San Pedro either as larvae through tidal currents or by adult migration. Therefore, the intertidal area here studied could be at the limit of the ecological distribution of this bivalve endosymbiont in the Cádiz Bay region.
The absence of juveniles inside the studied hosts suggests that this phase may be free-living and that the colonization of S. plana occurs during the benthic phase of the life cycle of O. okupa sp. nov., whose adults are able to move up along Río San Pedro with tides. However, neither juveniles, nor adults have been found in the sediments surrounding the studied areas (P. Drake, per. observ.), likely because they may be quite rare.
The possible life cycle of O. okupa sp. nov. may thus consist of 1) a planktonic larval phase settling on soft bottoms, 2) free-living juveniles, and 3) adults able to select (whenever possible) and enter the hosts at a given size (i.e., >1.6 mm wide according the present results). If so, phase 3 may involve thigmotaxis and the highly specific host entering behaviour described by Martin et al. (2015). In fact, a comparable life cycle was described for the polychaete Neanthes fucata (Savigny in Lamarck, 1818) in hermit crabs, by Gilpin-Brown (1969) off Plymouth. The planktotrophic larvae of this nereidid settle directly on soft bottoms. The juveniles live in tubes for several months feeding on detritus and small benthic animals exactly as many of their free-living relatives. Then, 4-month old worms start to develop the ability to recognize the presence of potential hosts by the substratum vibrations produced by the hermit crab legs bouncing on the sediment surface, which triggers a characteristic host-entering behaviour that allows the worms to crawl on the hermit crab shell following the shell spirals by thigmotaxis (Gilpin-Brown, 1969). This complex life cycle uses different mechanisms that characterize the free-living nereidids (such as thigmotaxis or mucus production) as specific adaptations to the commensal mode of life, while the worm itself has no relevant morphological adaptations. Therefore, in addition to the similarity of the hypothesized life cycle of O. okupa sp. nov., both species also share the lack of evident morphological adaptations (maybe except for the reduction of the central antennae in the hesionid, whose significance in terms of adaptation to the symbiotic mode of life remains unclear) and an equivalent, highly specific host entering behaviour.
The mechanism of host-recognition behaviour in O. okupa sp. nov. is currently unknown, and the presence of a host-factor has not been demonstrated (Martin et al., 2012, 2015). However, it is well known that the bivalves hosting O. okupa sp. nov. may alternate between direct water filtration and deposit feeding by tapping on the sediment surface with their inhalant siphons. Thus, we may hypothesize that the symbiont, like N. fucata, may recognize the presence of a potential host by the movements of the inhalant siphon. Despite aforementioned similarities between the known hosts of O. okupa sp. nov., there is no direct evidence of the symbiont entering into M. pellucida (or any other potential host) in a similar way as into S. plana.
Relationships between life–cycle and infestation characteristics
The infestation in O. okupa sp. nov., seemed to be connected with the reproduction. The months with lower prevalence (i.e., mid spring and summer) showed a markedly higher percentage of ripe females (>30%) (Fig. 4, 5; Table 9), reaching a 50% in August 2011, while the coldest month (i.e. October 2011 to February 2012) showed a high prevalence coupled with low percentages of ripe females (Fig. 4, 5; Table 9). The marked seasonal pattern found in the population of O. pugettensis (Johnson, 1901) infesting the starfish Patiria miniata (Brandt, 1835) at Dana Point (California) seemed also to be connected with the commensal reproductive dynamics (Lande and Reish, 1968). This species reached the highest (≥80%) and lowest (≤30%) prevalence in November-December and prior to the decrease of water temperature in summer, respectively. Its abundance was maximum in winter (2-3 worms per host) and minimum at mid-summer (<0.5 worms per host).
Despite sampling on different years is certainly required to infer regularities in the relationships between prevalence and life cycle in O. okupa sp. nov., these observations suggests that ripe females may have a reduced mobility (i.e., they tend to remain inside the host during their whole life), whereas males could be more mobile and thus leave their host, likely to increase fertilization success. Taking into account that O. okupa sp. nov. has a 1:1 regular distribution, the higher mobility attributed to males may contribute to an increase fertilization success by increasing the possibility of male/female partnership. Two possible mechanisms may explain this: 1) males may directly enter a host occupied by a ripe female, or 2) males may approach a host when females are releasing their sexual products. In both cases, we suggest that chemical cues may be involved. A similar behaviour was previously reported for B. seepensis, and, as mentioned above, also contributed to explain the lack of host-bivalve size relationships for adult males (Britayev et al., 2007). A similar situation was also reported for Haplosyllides floridana Augener, 1922, whose male stolons were never found inside the host sponge Neofibularia nolitangere (Duchassaing & Michelotti, 1864) and, conversely, were found free-swimming in the water column (Martin et al., 2009). Accordingly, the highest prevalence in the studied population of O. okupa sp. nov. occurred when both males and females occupied their respective hosts.
Despite this general pattern of reproduction vs. prevalence, O. okupa sp. nov. seems to reproduce actively during the whole year, but with an increasing effort during spring-summer and a higher intensity in summer. The absence of ripe females in April 2012 contrasted with the almost 40% found in 2011. However, ripe females occurred both before and after April 2012, allowing us to suggest that its absence in April 2012 could have been biased by the low number of symbionts found in this month (Fig. 4; Table 9). Persistent high temperatures can stimulate oocyte growth, subsequently causing the advancement of reproductive period in polychaetes, as reported for the Mediterranean populations of Eupolymnia nebulosa (Montagu, 1819) (Cha et al., 1997). However, our data does not allow assessing whether environmental constraints such as differences in temperature could affect the reproductive cycle of O. okupa sp. nov. during the studied period.
Current knowledge on hesionid symbionts
Thirty out of 170 currently known hesionid species live as commensals of other invertebrates (Martin and Britayev, 1998; Miller and Wolf, 2008; De Assis et al., 2012; Martin et al., 2012, 2015; Britayev et al., 2013; Chim et al., 2013), representing around 18% of all the known hesionid species and about 6% of the known symbiotic polychaetes (Table 10). Symbiosis seems to be restricted to the clade Ophiodrominae, which includes the genus Oxydromus. It has multiple origins within the family Hesionidae because Oxydromus and Gyptis, two of its most representative genera with symbiotic species, are not closely related phyllogenetically (Ruta, et al., 2007). Moreover, both are species-rich genera and there is no evidence indicating whether commensalism arose once or multiple times within each of them.
Commensal hesionids are involved in about 65 different associations (Table 10). Except for the polyxenous species of Oxydromus, such as for O. flexuosus (Delle Chiaje, 1827) and O. pugettensis (Johnson, 1901) with up to 10 and 11 hosts, respectively, most symbiotic hesionids are monoxoneus, occurring in only one (15 species) and two (6 species) hosts (Table 10). Hesionid hosts include species from very different taxonomic groups, a variety among symbiotic polychaetes only comparable to that of polynoids (Martin and Britayev, 1998). The most common hosts are, however, echinoderms (particularly starfishes and sea urchins) and polychaetes (Table 10).
Only one genus, Oxydromus, includes symbiotic species living in association with bivalves. These are O. pugettensis, O. humesi and O. okupa sp. nov. Although nothing is known on the relationships between O. pugettensis and its bivalve host, the polychaete seems to be able to detect at a certain distance the presence of at least two of its host starfishes, P. miniata and Luidia foliolata (Grube, 1866) (Davenport et al., 1960). Also there are some indications of mutualistic behaviour in their relationships with one of its echinoid hosts, the sand dollar Clypeaster humilis (Leske, 1778) (Storch and Niggemann, 1967). The existing analyses of the behaviour of O. okupa sp. nov. in experimental conditions do not prove the existence of a host-factor in its relationships with S. plana, like in the case of O. pugettensis and its host starfishes. In turn, the species shows an elaborated and complex host-entering behaviour, which leads the worm to enter inside the host bivalve mainly through the inhalant siphon (Martin et al., 2015). Moreover, the presence of O. okupa sp. nov. caused a significant reduction in the soft-body biomass of the infested hosts, compared to the non-infested ones, which may imply affectation of the host’s metabolism according to Bierbaum and Ferson (1986) thus leading to a relationship closer to parasitism (Martin et al., 2012). Such a negative influence has been previously reported for other symbiotic polychaetes living in the mantle cavity of bivalves, such as the deep-sea hydrothermal vent polynoids B. seepensis (Britayev et al., 2007). However, contrary to B. seepensis, in the case of O. okupa sp. nov. no damages in the tissues of S. plana were observed (Martin et al., 2012). In B. seepensis, tissue damages were considered as a secondary effect of the worm’s feeding inside the host caused by powerful jaws of the polynoid, and not a voluntary ingestion. Oxydromus okupa sp. nov. lacks jaws so that, even in the case of having a feeding mode similar to that B. seepensis, the hesionid seems to be able to avoid causing involuntary damage to the host tissues.
Despite the overall growing knowledge on symbiotic hesionids, O. okupa sp. nov. is probably the best known representative of the group to date. Nevertheless, further studies are required to complete the knowledge on this species, which becomes apparent if we compare it with O. humesi, with which it shares a similar morphology and a closely related host that is several thousands of kilometres away from Cadiz Bay. In other words, the present study is just another brick in the wall, which hopefully will encourage further research on the complex relationships between the symbiotic species of Oxydromus and their hosts.