Does Polyxenous Symbiosis Promote Sympatric Divergence? a Morphometric and Phylogeographic Approach Based on Oxydromus Okupa (Annelida, Polychaeta, Hesionidae)
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Contributions to Zoology (2019) 1-29 CTOZ brill.com/ctoz Does polyxenous symbiosis promote sympatric divergence? A morphometric and phylogeographic approach based on Oxydromus okupa (Annelida, Polychaeta, Hesionidae) Miguel A. Meca Centre d’Estudis Avançats de Blanes (CEAB-CSIC), Carrer d’Accés a la Cala Sant Francesc 14, 17300 Blanes (Girona), Catalunya, Spain [email protected] Pilar Drake Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), Avenida República Saharaui 2, Puerto Real 11519, Cádiz, Spain Daniel Martin Centre d’Estudis Avançats de Blanes (CEAB-CSIC), Carrer d’Accés a la Cala Sant Francesc 14, 17300 Blanes (Girona), Catalunya, Spain Abstract The polychaete Oxydromus okupa lives in association with the bivalves Scrobicularia plana and Maco- mopsis pellucida in the intertidal of Río San Pedro (CI = Cádiz Intertidal) and adjacent to CHipiona (CH) harbour, and in the subtidal of the Bay of Cádiz (CS = Cádiz Subtidal). We analyse these populations morphometrically, ecologically (including infestation characteristics) and genetically (intertidal popula- tions, 16S and ITS-1 genes). We consider “host”, “environment” and the combined “host and environment” as possible factors of interpopulation variability. Morphometry revealed three well-defined clusters for CI, CH and CS, showing intergroup phenotypic differences ranging from 35 to 50%. Hosts shell lengths ranged between 26 and 36 mm for S. plana and 20 and 28 mm for M. pellucida. The infestation of small M. pellucida by juvenile O. okupa suggests they show an active size segregation behaviour. The intertidal seems to be less favourable (infestation rate <25% vs. up to 65% in the subtidal), and did not show recent bottleneck events. Overall, CI and CH were genetically homogeneous, but showed a significant divergence (one dominant haplotype in each host species), suggesting host shift as being a soft barrier to gene flow. Most characters related with host-entering varied among populations, suggesting symbiotic behaviour to play a key role in reducing panmixia and leading to the initial phases of a speciation process in sympatric symbiotic populations. Polyxeny and symbiotic behaviour in O. okupa seem thus to be underlying mecha- nisms contributing to its great phenotypic variety, marked ecological differences, and genetic divergence. © meca et al., 2019 | doi:10.1163/18759866-20191403 This is an open access article distributed under the terms of the prevailing cc-by license at the time of publication. 2 10.1163/18759866-20191403 | Meca et al. Keywords Behaviour – Bivalvia – demography – evolution – Macomopsis – morphology – panmixia – Scrobicularia – symbiosis – sympatry 1 Introduction environmental preferences affect morphol- ogy, gene flow and demography, ultimately Sympatric populations (i.e., conspecific pop- attempting to clarify diversification mecha- ulations with genetically based phenotypic nisms in marine invertebrate symbionts. The differences that coexist spatially) are of in- symbiotic hesionid polychaete Oxydromus terest in evolutionary biology, as they hold humesi (Pettibone, 1961) was described as be- the potential of becoming a first step toward longing to Parasyllidea by Pettibone (1961), sympatric speciation. They may also be more based on a single population living in asso- common than hitherto thought, since they ciation with the tellinid bivalve Austroma- may have passed unnoticed using the number coma nymphalis (Lamarck, 1818) in mangrove of loci typical of the pre-genomics era (Jorde swamps in the Republic of Congo (Martin et et al., 2018). The rich cryptic diversity of poly- al., 2015). Two populations were later report- chaetes (Nygren, 2014) triggered an increas- ed as symbionts of bivalves, i.e., the semelid ing number of studies focusing on sympatric Scrobicularia plana (da Costa, 1778) in the populations (e.g., Nygren et al., 2010; Nygren & intertidal of Río San Pedro and the tellinid Pleijel, 2011; Zanol et al., 2016; Styan et al., 2017; Macomopsis pellucida (Spengler, 1798) in the Nygren et al., 2018). In the particular case of subtidal of the Bay of Cádiz, both at the south- symbiotic polychaetes, however, polyxenous ern Atlantic coast of the Iberian Peninsula species (i.e., symbiotic species inhabiting dif- (Martin et al., 2012, 2015). The Iberian popu- ferent hosts) appear to be less pervasive than lations were finally described as belonging to monoxenous ones (i.e., symbionts inhabiting a new species based on morphometric analy- a single host) (Martin & Britayev, 1998, 2018), ses, i.e., Oxydromus okupa Martin, Meca & Gil while they are the most likely candidates for in Martin et al. (2017). hidden cryptic speciation among sympatric The population associated with S. plana populations. To date, the only known study on was studied in detail in terms of behaviour, the phylogeography of symbiotic polychaetes infestation characteristics, and life cycle comprised the monoxenous Mediterranean (Martin et al., 2015, 2017). It showed a regular Ophryotrocha mediterranea Martin, Abelló distribution (i.e., a single symbiont individual & Cartes, 1991 and the polyxenous Atlantic- per host), intra-specific aggression, a complex Mediterranean Iphitime cuenoti Fauvel, 1914 host-entering behaviour, and low (usually (Lattig et al., 2016). This study showed that po- <5%) and seasonally fluctuating infestations lyxeny did not represent a biological barrier that were closely related with the reproduc- to gene flow among sympatric populations of tive cycle (e.g., males leaving their hosts dur- the latter species that inhabit four species of ing spring/summer, likely for fertilization brachyuran crabs. purposes). The life cycle postulated for O. ok- The present study concerns a different po- upa consisted on 1) a planktonic larval phase lyxenous relationship under sympatric condi- settling on soft bottoms when competent, tions. The aims are to assess whether host and 2) free-living juveniles, and 3) adults able to Diversification mechanisms | 10.1163/18759866-20191403 3 select (whenever possible) and enter the hosts (family Tellinidae) in the intertidal of Micaela at a given size. Beach adjacent to Chipiona harbour; CS In this study, we report a third popula- (Cádiz Subtidal) with M. pellucida in the sub- tion of O. okupa, recently discovered living tidal zone at the outlet of Río San Pedro in the intertidally in association with M. pellucida Bay of Cádiz (fig. 1). at Micaela Beach, adjacent to the harbour of Materials from previous sampling belong- Chipiona, ca. 40 km north-west of the Bay of ing to CS (January 2013) and CI (April, June, Cádiz. We take this opportunity to assess the July, August, September, October, November possible existence of population-level differ- and December 2011 and January, February, ences in morphology, gene flow, and demog- March, April and May 2012) (Martin et al., raphy. We consider two factors, host (S. plana 2017) were used for morphometric analyses. vs. M. pellucida) and environment (subtidal Additional specimens were collected in May vs. intertidal), as well as their combined effect 2016 by hand from the sediment during low (intertidal S. plana vs. subtidal M. pellucida). tide in CH (fig. 1). Specimens for morphome- Therefore, our aims are: (1) to check for try were relaxed in 7.2% magnesium chloride variations in morphology (based on mor- in distilled water, preserved in 4% seawater/ phometry) and ecology (based on population- formalin solution for a few days, rinsed in size structure and infestation rates); (2) to test fresh water, and transferred to 70% ethanol. the possible influence of host shift in gene For genetic purposes, additional specimens flow, based on two fast-evolving neutral mark- were collected on September 2017 (CH) and ers (i.e., 16S and ITS-1) that proved to be useful on September and November 2017 and May in assessing relationships amongst conspecif- and June 2018 (CI), and were preserved direct- ic populations in polychaetes (Nygren, 2014); ly in 100% ethanol. Attempts to obtain DNA- and (3) to analyse the symbiont demographic grade materials from CS in October 2017 and history by mismatch distribution, estimates June 2018 failed, likely due to changes in the of genetic diversity, and neutrality evolu- bottom configuration in this highly dynamic tion tests (based on the two aforementioned region, which prevented finding any speci- genes). Furthermore, we are providing new men in the previously surveyed area. As a re- sequence information on Hesionidae. Despite sult, 38 symbionts (19 from CH and 19 from CI) some recent papers (e.g., Pleijel et al., 2011, were available for genetics. 2012; Jimi et al., 2018; Rouse et al., 2018), this information is still very scarce in relation to 2.2 Morphometry the high species diversity of this family, par- Morphometric analyses were based on 68 ticularly for the symbiotic species. specimens (20 from CH, 23 CS, 25 from CI), all of them fixed in formalin and preserved in al- cohol (as there may be significant differences 2 Material and methods in appendage measurements according to the fixation method). The following measure- 2.1 Sampling ments were selected (following Martin et al., Specimens of O. okupa were collected from 2017; fig. 2): worm length (WL, μm), number of three populations of bivalve hosts in the Gulf segments (NS), worm width without parapo- of Cádiz: CI (Cádiz Intertidal) with S. plana dium (WWP, μm), length of dorsal lobe (DL, (family Semelidae) in the intertidal of Río μm), length of dorsal cirrophore (DCP, μm), San Pedro;