Aphrodita minuta Fabricius, 1780: 314 – 315

Aphrodita minuta Fabricius, 1780: 314 – 315 .

? Pholoe minuta Augener, 1928: 673 View in CoL .

Neotype: Paamiut, south of airfield, 62 ° 0.551 0 N, 49 ° 40.555 0 W, 10 June 2009, depth 2 m, sand and mud, formalin: complete specimen (ZMUC-POL-2405).

Non-type material: North Atlantic Ocean , Western Greenland: Pakitsup ilordlia, near meltwater runoff, 69 ° 29.776 N 50 ° 38.520 W, 06-Jun-09, depth 6 – 8 m, formalin: 25 af ( ZMH-P 27771 ) GoogleMaps , 4 af ( ZSRO-P2400 ) , depth 15 m, formalin: 18 af ( ZSRO-P2397 ) , 96% ethanol: 8 complete, 7 af ( SMF 24085 About SMF ) , 4 complete, 2 af ( ZSRO-P2385 ) ; Kangersuneq bay , N of Rodebay, 69 ° 20.624N 50 ° 54.985W, 06-Jun-09, depth 6 – 8 m, formalin: 16 af ( ZMH-P 27775 ) GoogleMaps , 5 af ( SMF 24092 About SMF ) , 1 af ( ZSRO-P2394 ) , 96% ethanol: 2 complete, 2 af ( ZSRO-P2384 ) ; Ilulissat , N of airfield, 69 ° 15.249 N 51 ° 2.822 W, 05-Jun-09, eulittoral, formalin: 5 af ( ZSRO-P2390 ) GoogleMaps , 35 af ( ZSRO-P2398 ) , 96% ethanol: 6 complete, 18 af ( ZSRO-P2382 ) , S of airfield, 69 ° 14.137 N 51 ° 4.2494 W, 08-Jun-09, eulittoral, mud, influenced by meltwater, formalin: 9 complete, 2 af ( SMF 24091 About SMF ) GoogleMaps ; Aasiaat (Egedesminde), 68 ° 42.634 N 52 ° 52.310 W, year 1890, formalin: 1 complete (ZMUC-POL-2362); Paamiut, S of airfield, 62 ° 0.709 N 49 ° 41.208 W, 11-Jun-09, eulittoral, sandy mud, formalin: 4 af ( ZSRO-P2388 ) GoogleMaps , 12-Jun-09, eulittoral, sand between stones, gravel, Fucus , formalin: 19 af ( ZMH-P 27770 ) , 96% ethanol: 1 complete, 3 af ( ZSRO-P2386 ) ; 62 ° 0.623 N 49 ° 41.075 W, 12-Jun-09, eulittoral, sand, Arenicola , formalin: 11 complete, 4 af ( ZMH-P 27777 ) GoogleMaps ; 62 ° 0.623 N 49 ° 41.208 W, 12-Jun- 09, mixed sediment, 96% ethanol: 7 af ( ZMH-P 27769 ) GoogleMaps ; 62 ° 0.551 N 49 ° 40.555 W, 10-Jun-09, depth 2 m, sand and mud, formalin: 7 complete, 6 af ( ZSRO-P2395 ) GoogleMaps , 96% ethanol: 2 complete, 3 af ( ZMHP 27768 ) ; near Paamiut , Iluilarssuk ^, type locality, 61 ° 50.059N 49 ° 25.183W, 14-Jun-09, eulittoral, rock pool influenced by meltwater, formalin: 1 complete ( ZSRO-P2392 ) GoogleMaps , 96% ethanol: 1 complete ( ZSROP2387 ) .

Diagnosis: Specimens brownish or greenish, with partially black-bordered light-yellow spot in the centre of the elytra; mid-dorsum usually completely covered by elytra or sometimes narrow gap left uncovered along the dorsal midline; elytra undulated, elytral papillae usually not articulated; facial tubercle absent; lateral antenna absent; neurochaetae heterogomph compound chaetae with finely serrated blades.

Description: Neotype complete specimen (without anal cirri) with 43 chaetigers, about 8.5 mm in length and 1.5 mm wide. Examined specimens were 4 – 18 mm long and 0.8 – 1.7 mm wide; large specimen with 56 chaetigers, 16 mm long, 1.7 mm wide. Body short, linear, depressed; ventral surface papillate with longitudinal groove. Mid-dorsum usually completely covered by elytra, sometimes narrow gap left uncovered along the dorsal midline ( Figs 5A, C, 7A View Figure 7 ); often elytra slightly ruffled ( Fig. 6A, H View Figure 6 ). First pair of elytra rounded ( Figs 5A, D, 8C View Figure 8 ), in succeeding segments reniform ( Figs 5B, I, J, 8D View Figure 8 ), in posterior segments becoming rounded again; segments without elytra with nodular lobes in the position of elytrophores; each elytron with short marginal papillae, also some papillae distributed over the dorsal elytral surface, in particular in anterior elytra and close to the posterior and lateral margins ( Figs 5A, D, I, J, 8C – D View Figure 8 ); elytral papillae usually not articulated, sometimes appearing pseudoarticulated ( Figs 6G, I View Figure 6 , 8C, D View Figure 8 ); elytral surface often pigmented (see below, under pigmentation; Fig. 7A View Figure 7 ).

Prostomium with smooth, distally tapering median antenna without articulations ( Figs 5A, B, 7B View Figure 7 ); lateral antenna absent; with two pairs of closely set, black eyes, sometimes anterior and posterior pair of eyes fused ( Figs 5A, B, 7B View Figure 7 ). Facial tubercle absent ( Figs 6A, C View Figure 6 , 7B View Figure 7 ). Tentacular segment achaetous, with two pairs of tentacular cirri rising from a tentaculophore, dorsal and ventral tentacular cirrus cirriform with few simple papillae ( Fig. 6C View Figure 6 ). Palps massive, tapering ( Figs 5A, B, 6C View Figure 6 ).

Parapodia biramous, proximoventrally with numerous simple short papillae ( Figs 5E, K, 8A, B View Figure 8 ); notopodium short, of conical shape at the end, without distinct terminal papillae, simple papillae along its anterior and posterior edge arranged in an irregular row ( Figs 5E, K, 6D View Figure 6 , 8A, B View Figure 8 ); neuropodium tapering, longer than notopodium, with numerous simple papillae distributed mainly anteriorly and ventrally over the surface, few inconspicous terminal papillae present ( Figs 5E, K, 6D View Figure 6 , 8A, B View Figure 8 ); cirriform ventral cirrus present on neuropodia, ventral cirrus at first chaetiger (so-called buccal cirrus) anteriorly oriented and slightly larger than on other chaetigers, otherwise laterally oriented. Both podial lobes bearing single stout aciculae that penetrate epidermis ( Fig. 5E, K); notopodium with long, spinous capillaries and short, geniculate, spinous capillaries ( Figs 5G, H, 6E, J View Figure 6 ); neurochaetae compound falcigerous, heterogomph chaetae with fine serrations of the blade and few serrations at the tip of the shaft ( Figs 5F, 6F View Figure 6 , 7C View Figure 7 ); blade length variable, usually longest in superior neurochaetae and shortest in inferior neurochaetae.

Pygidium with pair of cirriform anal cirri ( Fig. 5C), anus dorsal.

Pigmentation: Specimens with greenish to brownish pigment at the base of the antenna and tentacular cirri, anteriorly on the dorsum, on the ventral surface, and around the elytrophores; elytra of the same colour, with partially black-bordered light-yellow spot in the centre, where the elytron is attached below to the elytrophore ( Fig. 7A View Figure 7 ); sometimes with ‘rusty’, orange-coloured cover on the elytra that can be brushed off ( Fig. 7A View Figure 7 ). Pigment preserves well in 96% ethanol, but degrades in specimens originally preserved in formalin and later kept in 70% ethanol.

Biology: Specimens from station 31 (collected on 5 June 2009, north of Ilulissat) ovigerous. Eggs ovoid, 90 – 130 lm long, about 60 – 110 lm wide.

Geographical distribution (assessment based on molecular data, see following chapters of the present paper): West coast of Greenland, Hudson Bay ( Canada).

Remarks: The designated neotype was collected near Paamiut, not far from the exact position of the presumed type locality. Pholoe minuta is characterized by its greenish to brownish colour, with a light-yellow spot in the centre of the elytra, often ruffled elytra that usually cover the dorsum completely, the unarticulated elytral papillae, the absence of lateral antenna, the finely serrated blades of the compound neurochaetae, and the absence of a facial tubercle. The most recent taxonomic account of P. minuta , a literature review by Padovanni & Amaral (2013), listed some general morphological features of the species that are probably not sufficient for species identification. Comparing our results with characters listed by Padovanni & Amaral (2013) we can neither confirm that elytral papillae are distinctly annulated nor that they become larger towards the posterior region. The geographical distribution ranging from the Arctic to South Africa reported in the same publication seems doubtful. Currently the distributional range of the species can only be discussed by also considering information from genetic data (see Discussion).

GENETIC ANALYSES

Revision of archived sequence data

We retrieved all publicly available Pholoe sequences for COI, 16S, and 18S from GenBank and BOLD, and aligned those with our sequences obtained for P. longa and P. minuta from the species’ type localities. The COI alignment revealed that eight haplotypes deposited under P. minuta belong instead to P. longa , whereas 15 haplotypes tentatively archived under Pholoe sp. belong to P. minuta ( Table 2). The COI sequence of a specimen identified as P. baltica (Psp38, AY839585 View Materials ; Wiklund et al., 2005) showed a closer relationship to P. pallida (Psp39, AY894318 View Materials ; Struck et al., 2005) than to other haplotypes identified as P. baltica by Carr et al. (2011) and Hardy et al. (2011). Also considering clade affiliation (see below), we suggest referring to the latter as Pholoe sp. I and to Psp38 and Psp39 as Pholoe sp. IV for the present. Furthermore, the identities of 22 haplotypes belonging to at least five other Pholoe species (II, VI, VII, VIII, and IX) remain currently unknown ( Table 2). Likewise, the 16S and 18S sequences deposited for P. baltica and P. pallida ( Struck et al., 2005; Wiklund et al., 2005; Worsaae et al., 2005; Norlinder et al., 2012) should preferably be referred to as Pholoe sp. until the identities of these species are reliably fixed (see below).

Phylogenetic relationships in mitochondrial proteincoding COI

For the COI alignment, all available Pholoe sequences deposited in GenBank and BOLD ( Table 2) were combined with the data obtained in the present study. This resulted in 588 aligned nucleotides from 142 individuals (65 haplotypes), and included sequences from eight sites. For the alignment with the outgroup taxa the sequences were shortened to 576 nucleotides. Intraspecific distances in P. longa and P. minuta were 1.2 and 1.6%, respectively, and between those two taxa the distance ranged from 26.3 to 28.2% ( Table 3). The lowest interspecific distances (11.9%) were found between P. longa and two GenBank sequences of ‘ P. baltica ’ ( AY839585 View Materials ; Wiklund et al., 2005) and ‘ P. pallida ’ ( AY894318 View Materials ; Struck et al., 2005), and the highest (30.2%) were found between P. minuta and haplotype Psp8 (clade VIII; Table 3), an unidentified individual from St Andrews ( Canada; HQ024191 View Materials ; Carr et al., 2011). Phylogenetic analysis revealed 204 parsimony-informative characters in the alignment (35%), and 180 equally parsimonious trees (623 evolutionary steps) were used for tree reconstruction. Tree reconstructions ( Fig. 9) identified nine clades supported by significant consensus indices: clade I comprises haplotypes identified as ‘ P. baltica ’ ( Carr et al., 2011; Hardy et al., 2011), originating from sampling sites in Russia and North America. Clades II and IX include unidentified haplotypes from North American sites ( Carr et al., 2011), and are sisters to clades III and IV. Clade III combines the Greenlandic P. longa haplotypes of the present study with individuals that were identified as P. minuta from North America ( Carr et al., 2011). Two of the haplotypes found from Greenlandic populations were identical to haplotypes from Churchill ( Canada; Carr et al., 2011), whereas the other two were exclusive to Kangersuneq Bay ( Greenland). The sister clade IV consists of ‘ P. baltica ’ ( AY839585 View Materials ; Wiklund et al., 2005) and ‘ P. pallida ’ haplotypes from European populations ( AY894318 View Materials ; Struck et al., 2005). In clade V, the largest group, Greenlandic P. minuta haplotypes are combined with unidentified haplotypes from Churchill ( Canada; Carr et al., 2011). Four new haplotypes for P. minuta were found in the Greenlandic populations of Ilulissat and Paamiut; three individuals from these sites had the same and very common haplotype HH9. Clades VI, VII, and VIII consist of haplotypes identified as Pholoe sp. from the sampling sites at Churchill (clade VI) and St Andrews (clades VII and VIII) in Canada ( Carr et al., 2011).

The amplified COI fragment translated into 196 amino acids, and Pholoe species were divided into three clades based on four variable residues ( Fig. 9). One clade is composed of ‘ P. baltica ’ based on Ser at positions 88 and 108, Ala at position 107, and Ile at position 164. In the second clade, P. longa was grouped with GenBank ‘ P. pallida ’ and ‘ P. baltica ’, and with an unknown species of clade II. This clade differs from the ‘ P. baltica ’ clade by an Ala (position 108) and Val (position 164). Pholoe minuta is included in a third clade together with unknown species of nucleotide clades VI, VII, and VIII, based on Ala (position 88), Ser (positions 107 and 108), and Ile (position 164). Outgroup taxa, representing the ancestral state, had Ser at position 88, Ser or Ala at position 107 and 108, and Ile at position 164. Analysis of the partial COI sequences for selection revealed only negatively selected sites in Pholoe .

Genetic diversity in mitochondrial 16S

The alignment of 16S combined 29 sequences with 316 bp from five species ( P. minuta , P. longa , ‘ P. baltica ’, ‘ P. pallida ’, and Pholoe sp. ), resulting in 11 haplotypes. After alignment of Pholoe spp. with the outgroup, the alignment contained 343 bp. Pholoe minuta is represented by three haplotypes (HH16 – 19; Table 1; p-distance 0.3 – 0.6 %; Table 3), Species ID

Species ID (according to Sampling location

Haplotype (according to original GenBank/BOLD according to code present study) reference) accession no. reference Reference

COI

Pm1 Pholoe longa (III) Pholoe minuta GU 672255 Churchill ( Canada) Carr et al. (2011) Pm2 Pholoe longa (III) Pholoe minuta HQ 024187 St Andrews ( Canada) Carr et al. (2011) Pm3 Pholoe longa (III) Pholoe minuta GU 672371 Churchill ( Canada) Carr et al. (2011) Pm4 Pholoe longa (III) Pholoe minuta GU 672311 Churchill ( Canada) Carr et al. (2011) Pm5 Pholoe longa (III) Pholoe minuta HM 473782 Bering Sea ( USA) Carr et al. (2011) Pm6 Pholoe longa (III) Pholoe minuta GU 672206 Churchill ( Canada) Carr et al. (2011) Pm7 Pholoe longa (III) Pholoe minuta HQ 024186 St Andrews ( Canada) Carr et al. (2011) Pm8 Pholoe longa (III) Pholoe minuta HM 473781 Bering Sea ( USA) Carr et al. (2011) Psp1 Pholoe sp. (VII) Pholoe sp. HQ024188 View Materials St Andrews ( Canada) Carr et al. (2011) Psp2 Pholoe sp. (IX) Pholoe sp. HM473783 View Materials Bering Sea ( USA) Carr et al. (2011) Psp3 Pholoe sp. (VII) Pholoe sp. HQ024189 View Materials St Andrews ( Canada) Carr et al. (2011) Psp4 Pholoe sp. (VII) Pholoe sp. HQ024190 View Materials St Andrews ( Canada) Carr et al. (2011) Psp5 Pholoe sp. (II) Pholoe sp. HQ932539 View Materials Bamfield ( Canada) Carr et al. (2011) Psp6 Pholoe sp. (II) Pholoe sp. HQ932568 View Materials Haida Gwaii ( Canada) Carr et al. (2011) Psp7 Pholoe sp. (II) Pholoe sp. HM473560 View Materials Bamfield ( Canada) Carr et al. (2011) Psp8 Pholoe sp. (VIII) Pholoe sp. HQ024191 View Materials St Andrews ( Canada) Carr et al. (2011) Psp9 Pholoe minuta (V) Pholoe sp. GU672236 View Materials Churchill ( Canada) Carr et al. (2011) Psp10 Pholoe minuta (V) Pholoe sp. HQ023713 View Materials Churchill ( Canada) Carr et al. (2011) Psp11 Pholoe minuta (V) Pholoe sp. HQ938306 View Materials Churchill ( Canada) Carr et al. (2011) Psp12 Pholoe minuta (V) Pholoe sp. HQ938307 View Materials Churchill ( Canada) Carr et al. (2011) Psp13 Pholoe minuta (V) Pholoe sp. HQ023716 View Materials Churchill ( Canada) Carr et al. (2011) Psp14 Pholoe minuta (V) Pholoe sp. HQ023726 View Materials Churchill ( Canada) Carr et al. (2011) Psp15 Pholoe minuta (V) Pholoe sp. GU672247 View Materials Churchill ( Canada) Carr et al. (2011) Psp16 Pholoe minuta (V) Pholoe sp. HQ023718 View Materials Churchill ( Canada) Carr et al. (2011) Psp17 Pholoe minuta (V) Pholoe sp. GU672186 View Materials Churchill ( Canada) Carr et al. (2011) Psp18 Pholoe minuta (V) Pholoe sp. N /A Churchill ( Canada) Carr et al. (2011) Psp19 Pholoe minuta (V) Pholoe sp. HQ023723 View Materials Churchill ( Canada) Carr et al. (2011) Psp20 Pholoe minuta (V) Pholoe sp. HQ023728 View Materials Churchill ( Canada) Carr et al. (2011) Psp21 Pholoe minuta (V) Pholoe sp. HQ023717 View Materials Churchill ( Canada) Carr et al. (2011) Psp22 Pholoe minuta (V) Pholoe sp. HQ023722 View Materials Churchill ( Canada) Carr et al. (2011) Psp23 Pholoe minuta (V) Pholoe sp. HQ023714 View Materials Churchill ( Canada) Carr et al. (2011) Psp24 Pholoe sp. (VI) Pholoe sp. HQ023712 View Materials Churchill ( Canada) Carr et al. (2011) Psp25 Pholoe sp. (VI) Pholoe sp. GU672136 View Materials Churchill ( Canada) Carr et al. (2011) Psp26 Pholoe sp. (VI) Pholoe sp. GU672203 View Materials Churchill ( Canada) Carr et al. (2011) Psp27 Pholoe sp. (VI) Pholoe sp. HQ023707 View Materials Churchill ( Canada) Carr et al. (2011) Psp28 Pholoe sp. (VI) Pholoe sp. GU672211 View Materials Churchill ( Canada) Carr et al. (2011) Psp29 Pholoe sp. (VI) Pholoe sp. HQ023702 View Materials Churchill ( Canada) Carr et al. (2011) Psp30 Pholoe sp. (VI) Pholoe sp. HQ023692 View Materials Churchill ( Canada) Carr et al. (2011) Psp31 Pholoe sp. (VI) Pholoe sp. GU672246 View Materials Churchill ( Canada) Carr et al. (2011) Psp32 Pholoe sp. (VI) Pholoe sp. HQ023697 View Materials Churchill ( Canada) Carr et al. (2011) Psp33 Pholoe sp. (VI) Pholoe sp. HQ023693 View Materials Churchill ( Canada) Carr et al. (2011) Psp34 Pholoe sp. (VI) Pholoe sp. HQ023701 View Materials Churchill ( Canada) Carr et al. (2011) Psp35 Pholoe sp. (VI) Pholoe sp. GU672210 View Materials Churchill ( Canada) Carr et al. (2011) Psp36 Pholoe sp. (VI) Pholoe sp. GU672248 View Materials Churchill ( Canada) Carr et al. (2011) Psp37 Pholoe sp. (VI) Pholoe sp. HQ023699 View Materials Churchill ( Canada) Carr et al. (2011) Psp38 Pholoe sp. (IV) Pholoe baltica AY 839585 Koster Area ( Sweden) Wiklund et al. (2005) Psp39 Pholoe sp. (IV) Pholoe pallida AY 894318 Trondheim ( Norway) Struck et al. (2005) Pb1 Pholoe sp. (I) Pholoe baltica GU 672253 Churchill ( Canada) Carr et al. (2011) Pb2 Pholoe sp. (I) Pholoe baltica HQ 561101 Churchill ( Canada) Carr et al. (2011) The following haplotypes were identical to haplotypes sampled in this study: Psp13 = HH9; Pm6 = HH7; and Pm3 = HH8. Roman numerals given in parentheses in the second column refer to the clades in Figure 9.

whereas all P. longa sequences had the same haplotype (HH18; Table 1). Pholoe minuta and P. longa differed by 13.1% from each other. Interestingly, the Pholoe sp. individual from the North Sea (HH10) had the same haplotype as ‘ P. pallida ’ from Norlin- der et al. (2012), but was by 15.9% different from the ‘ P. baltica ’ ( AY839585 View Materials ) of the same study. In the phylogenetic analyses, P. minuta was placed as a sister to a clade with specimens provisionally determined as Pholoe sp. , and P. longa was sister to a clade consisting of ‘ P. baltica ’ and Pholoe sp. sequences ( Fig. S1). The haplotype shared by HH10 and ‘ P. pallida ’ ( JN852912 View Materials ; Norlinder et al., 2012) was placed as a sister to the other clades.

Genetic diversity in nuclear 18S

The 18S alignment resulted in 462 nucleotides from 36 sequences of five species ( P. minuta , P. longa , ‘ P. baltica ’, ‘ P. pallida ’, and Pholoe sp. ). Only three haplotypes were found, with HH21 ( Table 1) being the most common 18S haplotype (N = 34). All P. minuta and P. longa sequences shared this haplotype, and thus no species-specific differences were found between both species. Furthermore, two ‘ P. baltica ’ ( AY176301 View Materials and AY839573 View Materials ) and four individuals assigned to Pholoe sp. from North Sea sites (this study) showed this haplotype. Sequences of ‘ P. pallida ’ ( AY894318 View Materials ) and HH20 ( Pholoe sp. ; Table 1) had between 0.3 and 0.6% (between three and six substitutions) difference to haplotype HH21, which comprised all other sequences.

Haplotype networks of COI

To picture the spatial distribution among Pholoe haplotypes and to detect the ancestral haplotypes, we constructed haplotype networks from COI sequences. The haplotype network reconstruction of P. longa ( Fig. 10A View Figure 10 ) included 37 sequences from seven populations. The mean number of polymorphic sites in P. longa was 3.1 2.3 ( Table 4). The results revealed HH8 as the central and ancestral haplotype; 65% (N = 24) of the sequenced individuals had this haplotype, and it was found in five populations from both North American and Greenland. In contrast to the P. minuta network, a geographic pattern was observed in the P. longa network ( Fig. 10A View Figure 10 ; Table S2). The haplotypes (Pm2, Pm7, and Pm8) from the Bering Sea (Alaska) and St Andrews (New Brunswick) were clustered together, and were separated from the other haplotypes by sharing one synapomorphy (position 297). Only Pm5 from the Bering Sea showed a closer relationship to haplotype