Introduction

The genus Prionospio Malmgren 1867 is one of the most diverse and speciose taxon within the Spionidae (Guggolz et al. 2018; Paterson et al. 2016). Prionospio is morphologically not well defined, even after several revisions and the erection of closely related subgenera and new genera in a Prionospio complex (Foster 1971; Maciolek 1985; Sigvaldadottir 1998; Wilson 1990). Currently, the Prionospio complex comprises the genera Aurospio Maciolek 1981, Laubieriellus Maciolek 1981a, Orthoprionospio Blake and Kudenov 1978, Prionospio Malmgren 1867, Streblospio Webster 1879, and Paraprionospio Caullery 1914. Most of these revisions were mainly based on shallow-water species, but Prionospio can also be regarded a typical deep-sea genus, often found in high abundances (Blake et al. 2018; Guggolz et al. 2018; Guggolz and Meißner pers. observations).

Despite the typical occurrence of Prionospio in deep-sea samples, the number of reported species is rather limited (Glover and Fauchald 2018: 29 species—http://www.marinespecies.org/deepsea/). Only a few generic characters are available to distinguish between the genera of the Prionospio complex; often, only the arrangement of the branchiae is important for the characterization of different subgenera and genera (Paterson et al. 2016). These characters seem to be sufficient to identify specimens in appropriate conditions, but the morphological identification of these soft-bodied annelids from deep-sea samples is often difficult. Due to their fragility, the majority of specimens from these depths are incomplete or damaged (Bogantes et al. 2018; Guggolz et al. 2018, 2019). For example, the genus Aurospio is mainly distinguished from Prionospio by the number of the branchiae and on which segment they are beginning (Sigvaldadóttir and Mackie 1993), but these appendages are often lost or damaged during sampling procedures.

Species of both genera, Prionospio and Aurospio, are reported to be widespread (Paterson et al. 2016) or even cosmopolitan (Mincks et al. 2009) (e.g., Aurospio dibranchiata Maciolek 1981). A wide dispersal potential in the abyssal has been reported for many benthic invertebrates (Etter et al. 2011; Linse and Schwabe 2018; Schüller and Ebbe 2007); however, these distribution patterns, based solely on morphological taxonomic identification, have to be treated with caution. Recent studies, employing molecular tools, often indicated a more complex scenario. Several of these presumably widespread species were found to be composed of a number geographically restricted and morphologically cryptic species (Bickford et al. 2007; Vrijenhoek 2009) or simply misidentified (Álvarez-Campos et al. 2017; Nygren et al. 2018; Sun et al. 2016). Hence, hypothesizing distribution patterns in the deep sea is still challenging, and integrative approaches, which combine morphological and molecular techniques, are essential to identify and delimit species (Glover et al. 2016; Hutchings and Kupriyanova 2018). One important aspect of the present study is to examine the diversity and the dispersal capacity of Prionospio and Aurospio in the Vema-Fracture-Zone (VFZ). Both genera, Prionospio and Aurospio, are supposed to have planktonic larvae and thus a potential for a widespread geographic distribution (Wilson 1991; Young 2003). We investigate the dispersal along the VFZ and test for barrier effect of the Mid-Atlantic Ridge (MAR) as this underwater mountain ridge is often postulated to represent a topographic barrier for distribution of benthic invertebrates (Bober et al. 2018; McClain et al. 2009; Priede et al. 2013). However, a barrier effect of the MAR on the spionid Laonice Malmgren 1867 has recently been rejected (Guggolz et al. 2019). The herein studied Prionospio and Aurospio will be another important step towards understanding the distribution patterns of species in the deep sea along the MAR. In addition, a potential pan-oceanic distribution is analyzed, by comparing DNA sequences of specimens from the VFZ (tropical Atlantic) with those of the Clarion-Clipperton Fracture Zone (CCZ) from the central Pacific.

Material and methods

Collection and identification of specimens

During the VEMA-Transit expedition in December 2014 to January 2015, 197 of the 332 analyzed specimens were collected from the tropical North Atlantic and the Puerto Rico Trench (Fig. 1: VFZ and PRT). Detailed information about sample treatment and sampling localities are described in Guggolz et al. (2018) and Devey (2015). Four areas were defined for samples from the Atlantic according to the geographical position as following: the eastern part of the Vema-Fracture Zone (eVFZ), extending eastwards from the MAR in the Cape Verde Basin; the western part of the Vema Fracture Zone (wVFZ), extending westwards from the MAR in the Demerara Basin; the Vema Transform Fault (VTF), located between these two areas in the MAR; the Puerto Rico Trench (PRT), located in the shallower part of the trench near Puerto Rico. Maximum distances within areas varied between 276 km (wVFZ) and 1298 km (eVFZ). The eastern-most and western-most studied sites were separated by 4610 km.

Fig. 1
figure 1

Map of the worldwide sampling localities. The Clarion Clipperton Fracture Zone (CCZ) in the Pacific, the eastern Vema Fracture zone (eVFZ–stars), the Vema Transform Fault (VTF–rectangular), the western Vema Fracture Zone (wVFZ–hexagon), and the Puerto Rico Trench (PRT)

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The Clarion-Clipperton Fracture Zone (CCZ) is a vast area (about 6 million km2) in the Equatorial Pacific Ocean with high commercial interest because of the presence of polymetallic nodules in the seabed between 4000 and 5000 m depth. The International Seabed Authority (ISA) is in charge of management of deep-sea mineral resources and of protection of marine environment in areas beyond national jurisdiction (Lodge et al. 2014). The ISA provides licenses to the contractors that intend to explore mineral deposits in, e.g., the CCZ. To get and keep an exploration contract for an area, the contractor is required to carry out surveys and fauna inventories (Lodge et al. 2014). Furthermore, the ISA administrates the regional environmental management plan across the CCZ, so-called Areas of Particular Environmental Interest (APEI). Sequences of 122 specimens morphologically assigned to Prionospio and Aurospio from eight exploration contract areas and one APEI were included (Fig. 1: the German exploration contract area ‘BGR’, Russia and Poland among other countries ‘IOM’, Belgium ‘GSR’, French ‘Ifremer’ & ‘Ifremer-2’, Singapore ‘OMS’, Britain UK-1 and the APEI-6). Of these, 53 specimens were collected on the two United Kingdom Seabed Resources Ltd. (UKSR) cruises AB01 and AB02 to the UK-1 exploration contract area stratum A and stratum B, the OMS contract area, and the APEI-6. Details on sampling methods are given in Glover et al. (2016). Maps and metadata from UK-1 stratum A have been published in earlier taxonomical work on macrofaunal material from this cruise (Dahlgren et al. 2016; A. Glover et al. 2016; Wiklund et al. submitted, 2017). In addition, 69 specimens morphologically assigned to Prionospio and Aurospio from BGR, IOM, GSR, and Ifremer were sampled using box-corers (0.25 m2) or epibenthic sledge during EcoResponse SO239 cruise on board of the RV Sonne in March/April 2015 funded by JPI Oceans framework (Martínez Arbizu and Haeckel 2015).

All specimens were sorted and identified at least to genus level (Prionospio or Aurospio) using dissecting and compound microscopes. Specimens have been deposited in the collection of the Center of Natural History (Universität Hamburg, Germany), Ifremer (France), and Natural History Museum London (Supplement 1).

The map of the sampling areas (Fig. 1) was created using ArcGIS 10.4.1 (www.esri.com).

DNA extraction, PCR amplification, sequencing, and alignment

For the VFZ specimens, one or two parapodia were dissected and transferred into 30 μl of 10% Chelex 100 solution in purified water, incubated for 30 min at 56 °C and 10 min at 99 °C. Polymerase chain reactions (PCR) were performed with a total volume of 15 μl consisting of 1.5 μl DNA extract, 7.5 μl AccuStart II PCR ToughMix (Quanta Bio, Germany), 0.6 μl of each primer (10 mmol), 0.3 μl of GelTrack loading dye (QuantaBio, Germany), and 4.8 μl Millipore H2O. Fragments of mitochondrial (16S) and nuclear (18S) rRNA genes were amplified (see Table 1 for list of primers) with initial denaturation step of 94 °C for 3 min, followed by 35 cycles of 30 s at 94 °C, 45 s at 43 °C, and 45 s at 72 °C, followed by a final elongation step for 5 min at 72 °C. Success of amplification was determined via gel electrophoresis on 1% agarose/TAE gel. For sequencing, 8 μl of the PCR products was purified using FastAP (1.6 μl; 1 U/μl) and Exonuclease I (0.8 μl; 20 U/μl) (Thermo Fisher Scientific, Germany) with an incubation time of 37 °C for 15 min followed by 15 min at 85 °C. For some of the specimens from the CCZ (BGR, IOM, GSR and Ifremer), DNA extractions were realized with NucleoSpin Tissue (Macherey-Nagel) kit and PCR amplifications as following into 25-μl mixtures, including 5 μl of Green GoTaq® Flexi Buffer (final concentration of 1×), 2.5 μl of MgCl2 solution (final concentration of 2.5 mM), 0.5 μl of PCR nucleotide mix (final concentration of 0.2 mM each dNTP), 9.875 μl of nuclease-free water, 2.5 μl of each primer (final concentration of 1 μM), 2 μl template DNA, and 0.125 U of GoTaq® G2 Flexi DNA Polymerase (Promega). The temperature profile was 95 °C/240 s, 94 °C/30 s, 52 °C/60 s, 72 °C/75 s *35 cycles, 72 °C/480 s, 4 °C. Purified PCR products were sent to Macrogen Europe, Inc. (Amsterdam-Zuidoost, Netherlands) for sequencing. The remaining specimens from the CCZ (APEI-6, OMS, UK-1) were extracted with DNeasy Blood and Tissue Kit (Qiagen) using a Hamilton Microlab STAR Robotic Workstation. PCR mixtures contained 1 μl of each primer (10 μM), 2 μl template DNA, and 21 μl of Red Taq DNA Polymerase 1.1× MasterMix (VWR) in a mixture of total 25 μl. The PCR amplification profile consisted of initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were purified using Millipore Multiscreen 96-well PCR Purification System, and sequencing was performed on an ABI 3730XL DNA Analyser (Applied Biosystems) at Natural History Museum Sequencing Facility, using the same primers as in the PCR reactions.

Table 1 Primers used in this study
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In total, 331 specimens were successfully sequenced for 16S rRNA and 63 specimens for 18S. Additionally, the mitochondrial COI gene and the nuclear 28S and ITS genes were tested with considerably worse success and therefore deliberately omitted. 18S is known to show some degree of divergence, even at species level in spionids, and thus successfully used for supporting mitochondrial markers (Guggolz et al. 2019; Meißner et al. 2014; Meißner and Blank 2009). Sequences were assembled and corrected with Geneious 6.1.8 (Kearse et al. 2012; http://www.geneious.com) and deposited in GenBank (for accession numbers, see Supplement Table 1). The obtained sequences of the different gene fragments were aligned separately using MAFFT (Katoh and Standley 2013; V 7.402) implemented with CIPRES Science Gateway V.3.3 (Miller et al. 2010; www.phylo.org).

Initial identification of species, phylogenetic analyses, and haplotype networks

To obtain a first estimation of the number of species present in our data set, the Automated Barcode Gap Discovery (Puillandre et al. 2012; ABGD) was conducted with 16S rRNA. The ABGD identifies potential barcoding gaps separating hypothetical species, which is based on the assumption that interspecific genetic distances are larger than intraspecific distances. The ABGD analysis was run on the web-based version of the software (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html). Pairwise uncorrected p-distances were used for the analyses (Table 2), which were calculated with MEGA7 (Kumar et al. 2016), including all available sequences for each gene, respectively. Standard settings for ABGD were kept, except for Pmin (0.005), the numbers of steps (100), and the relative gap width (X = 0.5). In the following, we will use the term lineages rather than species for the units delimited by ABGD, as not all of these might correspond to actual species. Additionally, the single locus tree-based species delimitation analysis GMYC (general mixed Yule coalescent; Pons et al. 2006) was used to support the results of the ABGD. For the GMYC analyses, an ultrametric tree was calculated with BEAST 2.4.6 (Bouckaert et al. 2010; www.phylo.org). Whether a given phylogenetic hypothesis can be rejected by the data is often not obvious from the tree topology, especially in cases where some branches are not significantly supported. We used the approximately unbiased test (Shimodaira 2002) as implemented in the program CONSEL (Shimodaira and Hasegawa 2001) to test the statistical significance of likelihood differences between the best ML topology and the ML topology, in which the genus Aurospio was constrained to be monophyletic. The constrained ML tree and the site-wise likelihoods associated with each topology (necessary for conducting the approximately unbiased test) were calculated with GARLI 2.0 (Zwickl 2006). Due to missing sequences of the 18S genes for a large part of the analyzed specimens and the very few mutations, only the phylogenetic analysis of the 16S rRNA gene is included. For the 16S rRNA analysis, different spionids that are not assigned to the Prionospio complex were chosen as outgroups (Supplement 1). Additionally, available 16S rRNA sequences of Prionospio and Aurospio from GenBank were included in the analyses (Supplement 1). Four chains were run for 5*107 generations, with sampling every 1200th generation, and discarding the first 25% as burn-in. Thus, the convergence chain runs were validated using TRACER v.1.7.1 (Rambaut et al. 2016).

One of the main aims of this study was to assess the distribution and dispersal potential of the studied species. Some species were found to be restricted to one of the investigated oceans (Prionospio sp. C, D, F, G, I, K, L, M, O, P and Aurospio sp. T; Figs. 2 and 3), but there are also several species found to have a pan-oceanic distribution (Prionospio sp. A, B, E, H, N and Aurospio cf. dibranchiata, sp. S; Figs. 2 and 3). Most of these species found in the Atlantic, as well as in the Pacific, show comparable patterns regarding the haplotype networks and thus of the distribution of their intraspecific genetic diversity. Identical haplotypes are shared between populations inhabiting both oceans (i.e., h9-16S, h35-16S, h95-16S, h119-16S; Fig. 3), and other haplotypes differ only on one or a few mutations in 16S rRNA between oceans. There are three possible scenarios explaining this remarkable finding: (1) shared haplotypes were already present in the ancestral population > 3 million years ago (O’Dea et al. 2016) before Atlantic and Pacific populations were separated by the Isthmus of Panama (vicariance), (2) historic gene flow between Atlantic and Pacific populations, and (3) recent and/or continuous gene flow between Atlantic and Pacific populations. In scenario (1), the observed distribution of haplotypes would be due to variance rather than dispersal and gene flow. Identical haplotypes would be ancestral and retained unchanged in both populations over 3 million years. This would imply very low substitution and speciation rates for these species as we have no reason to believe that the Atlantic and Pacific populations in question represent different species. In turn, this would imply very old ages for the studied genera and all other studied species pairs, as these species were separated by 5.1–32.0% uncorrected p-distance in 16S rRNA, which would require hundreds of millions of years to accumulate at this rate. We deem this rather unlikely and therefore favor scenarios (2) and (3).

Scenarios (2) and (3) both assume gene flow between Atlantic and Pacific populations of several species much later than the closure of the Isthmus of Panama. With the available data, it is not possible to assess the timing and frequency of gene flow events within each species. However, dispersal and gene flow rates are apparently too low to lead to population admixture between oceans. In several of the studied species, dispersal might have occurred only once, resulting in the colonization of the respectively other ocean, though repeated and on-going dispersal and gene flow is possible as well.

It is widely accepted that one of the main driving factors for such wide dispersal capacities of marine invertebrates are long-lived planktonic larval stages (Eckman 1996; Rex et al. 2005; Scheltema 1972; Schüller and Ebbe 2007; Yearsley and Sigwart 2011). Even if the specific type of larvae of the herein studied species is unknown, other species of Prionospio and Aurospio are reported to develop via planktonic larvae like other species of these genera (Blake et al. 2018; Mincks et al. 2009; Young 2003). The planktonic larval duration and dispersal distances are potentially higher in the deep sea, as the cold temperature and consequently reduced metabolic rates are identified as one of the main driving factors for extended larval stages (McClain and Hardy 2010; O’Connor et al. 2007). Considering the enormous distances between the CCZ and the VFZ, it seems highly unlikely that larvae drift all the way between the populations. It rather appears plausible that the distribution over such large geographical distances is linked to ocean currents, connecting different suitable habitat patches in a stepwise fashion (McClain and Hardy 2010; Rex and Etter 2010; Young et al. 2008). The direction of dispersal of Aurospio and Prionospio larvae remains unknown, as well as whether the species originated from the Pacific or the Atlantic. Probably, additional populations occur in the un-sampled regions between our study sites, which might have acted as step** stones. Alternatively, some of the studied species might have a continuous distribution linking the Atlantic and Pacific populations. Further comparison to populations from other localities would help to clarify these issues. The potential to find conspecific specimens in other deep-sea areas and another indication for the enormous distribution potential of some spionids is supported by specimens from the Crozet Islands (Antarctica) that were assigned to Prionospio sp. E. The Southern Ocean could also connect the Atlantic and the Pacific most likely with the eastwards flowing deep-water currents (Rahmstorf 2002; Stow et al. 2002).

A pan-oceanic distribution found for Aurospio and Prionospio species as evidenced from molecular data is so far unique for abyssal annelids, even if wide distribution ranges of polychaete species are not unusual (Böggemann 2016; Eilertsen et al. 2018; Georgieva et al. 2015; Guggolz et al. 2019; Meißner et al. 2016; Schüller and Hutchings 2012). However, Hutchings and Kupriyanova (2018) are particularly highlighting that reported “cosmopolitan” species should be treated with caution. For some species, wide distribution ranges have been based on misidentification or cryptic species and subsequently rejected using molecular marker (Álvarez-Campos et al. 2017; Nygren 2014; Nygren et al. 2018; Sun et al. 2016). The assumption that planktonic larvae in the deep sea are staying longer in the water column and transported via currents successfully over long distances raises the question of what the potential dispersal barrier restricting the distribution are. Life-history traits like larval behavior, larval mortality, and physiological tolerances for vertical movement and settlement in different habitats are probably important (Glover et al. 2001; McClain and Hardy 2010; Virgilio et al. 2009). It has been suggested that connectivity in the deep sea has often been associated with a common bathymetry rather than spatial vicinity (Glover et al. 2001). Thus, topographic barriers, like ridges, canyons, and rises, are supposed to influence distribution patterns (Eckman 1996; Guggolz et al. 2018; McClain and Hardy 2010; Won et al. 2003). Such a potential barrier is the MAR, dividing the Atlantic in eastern and western basins (Riehl et al. 2018). Some of the herein studied species (Prionospio sp. G, sp. L, sp. M and Aurospio cf. dibranchiata) occurring in the tropical Atlantic were found to be distributed across the MAR with wide distribution ranges of up to > 4000 km (Figs. 1 and 3) with no or only little genetic differentiation between populations. Several haplotypes were found to be identical (h29-16S, h59-16S, h61-16S, h69-16S, h119-16S, h121-16S; Fig. 3), indicating gene flow across the MAR. Even for species restricted to one side of the MAR (eVFZ: Prionospio sp. H, sp. P and Aurospio sp. S) or at least to the VTF (Prionspio sp. B, sp. E, sp. N, sp. O), shared haplotypes were found across hundreds of kilometers (47-16S, h9-16S, h22-16S, h87-16S; Figs. 1 and 3). These results are strongly refuting a barrier effect of the MAR for these Prionospio and Aurospio species. The dispersal potential across topographic barriers like the MAR and over large geographic distances was also reported recently for species of the spionid genus Laonice (Guggolz et al. 2019). The importance of dispersal via larvae is emphasized by distribution patterns found for brooding taxa like isopods, which exhibited very limited or no gene flow across the MAR (Bober et al. 2018; Brix et al. 2018).

In summary, the results of this study are expanding our knowledge of the diversity and distribution patterns of Aurospio and Prionospio in the deep sea. We could identify 21 lineages with some assigned to already described species (Aurospio foodbancsia, Aurospio cf. dibranchiata) and others remaining unknown at present. These other molecularly delineated potential species cannot be differentiated from described species, due to lack of sufficient morphological characters as most individuals were damaged and incomplete and because genetic data for most of the described species is lacking. The data do not support the monophyly for the two genera, which is highlighting the importance of molecular analyses additionally to morphological examinations to revise the Prionospio complex. A remarkable degree of pan-oceanic distribution in some of the species was shown, especially in the tropical Atlantic. The pan-oceanic distribution is indicating the potential of widespread distribution for Prionospio and Aurospio species, even over topographic barriers and should be kept in mind for further studies on distribution patterns in deep-sea polychaetes.