Molecular phylogeny of Squaliformes

On average, 200,000 of 352,605 possible basepairs, were sequenced per specimen (Additional file 1: Table S1). Characteristics of the raw dataset are given in Additional file 1: Table S2. Missing data were randomly distributed among specimens resulting in a large amount of incomplete sequences per captured locus and specimen.

MARE [33, 34] detected 174 phylogenetically informative loci in the raw dataset (Additional file 1: Figure S1). Re-blasting the full genome of C. milii against the 174 phylogenetically informative loci resulted in two potentially paraloguous loci (cds 1200 (unknown) and cds 1366 (LRP4)). Excluding these two loci and repeating the maximum likelihood analysis as described above did not affect the inferred tree topology.

Phylogenetic estimates presented herein provide a fully resolved and well-supported molecular hypothesis for the phylogeny of Squaliform sharks. The Maximum Likelihood trees as well as the Bayesian inferences resulting from different types of analyses carried out using RaxML [35] and PhyloBayes 3.3f [36, 37] were broadly congruent in topology except for the phylogenetic placement of Oxynotus. This taxon appears as sister taxon to all somniosid genera except for Somniosus in an analysis of all 1265 loci, but is nested among somniosid genera except for Somniosus in the analyses of the reduced dataset comprising 174 and 172 loci, respectively. The topology used for further analysis is summarized in Fig. 1, and is based on the 172 concatenated nucleotide loci that were selected through the MARE matrix reduction process and re-blasting analysis. The concatenated and aligned 172 nucleotide loci are deposited in the Dryad data repository [38] (Additional file 1: Tables S3 and S4, Figures S3 to S6).

Fig. 1 Maximum likelihood phylogenetic estimate of squalomorph sharks based on gene capture data of 172 nucleotide loci under a GTR + Gamma model using RAxML [35] partitioned into two sets, 1st and 2nd codon position as well as 3rd codon only. Analyzed specimens are listed in Additional file 1: Table S1. Nodes marked with black dots indicate 100 % bootstrap support and a posterior probability of 1 assessed in the Bayesian inference from the Phylobayes 3 analysis applying the CAT model [36, 37, 64]. Tree rooted midpoint, no outgroup defined. Full size image

This phylogenetic estimate reveals two major clades: the Squaliformes excluding Echinorhinidae and a clade containing Squatina, Pristiophoriformes, and Echinorhinus (Fig. 1). Within this clade, Echinorhinus is sister to Squatina and Pristiophoriformes. Results suggest that Echinorhinidae are not Squaliform sharks, but are the sister group to Angel- (Squatiniformes) and Saw sharks (Pristiophoriformes), as previously suggested by the analysis of mitochondrial data [21]. Therefore, Squaliformes form a monophyletic group only, if Echinorhinus is excluded. This study does not support results from [24], suggesting Echinorhinus being the sistergroup to the remaining Squaliform lineages. The node time estimation for the Echinorhinus lineage suggests an Upper Jurassic splitting of the extant Echinorhinus lineage and the Squatina plus Pristiophoriformes clade. This dates the Echinorhinus lineage older than anticipated from the fossil record, which reports the oldest echinorhinid fossil from the early Cretaceous (Hauterivian) of southeastern France [27], while the oldest squatinids already appear in the Upper Jurassic [25].

Within the Squaliform clade, the first split separates Squalidae from the remaining families Centrophoridae, Etmopteridae, Dalatiidae, Somniosidae, and Oxynotidae. The genera Squalus and Cirrhigaleus appear as sister taxa. Centrophoridae split from Etmopteridae, Dalatiidae, Somniosidae, and Oxynotidae, where genera Deania and Centrophorus are sister. Dalatiidae are sister to a clade comprising Etmopteridae, Somniosidae, and Oxynotidae. There are two clades within the dalatiids, one comprising the Isistius and Dalatias lineages, the other Squaliolus and Euprotomicrus. As shown in Fig. 1, Somniosidae sensu stricto form two clearly distinct lineages that are sister to each other, one containing the genus Somniosus (Fig. 1), the other lineage contains all other remaining somniosid genera. Oxynotidae cluster within Somniosidae (Fig. 1). Within Etmopteridae, Trigonognathus is sister to a clade comprising Aculeola and Centroscyllium. Etmopterus is sister to this previously described clade forming four distinct lineages representing the subclades described in [23].

Oxynotus is inferred to be nested within Somniosidae, rendering the family Somniosidae paraphyletic (Fig. 1) in the current study. This result is repeatedly recovered in phylogenetic estimates based on DNA sequence data (both mitochondrial and nuclear) [19–24]. Given the consistency of the inferences from molecular data, it would be interesting to see if any anatomical features also support the link between Oxynotidae and Somniosidae. Oxynotus clusters with a group of otherwise morphologically similar species of somniosids, i.e. along with Zameus, Centroselachus, Scymnodon, and Centroscymnus. Our molecular results show that all five genera are closely related (Fig. 1). This is especially evident when comparing intergeneric diversity within Somniosidae with the large intrageneric sequence differences evident within the genus Etmopterus (Fig. 1). Moreover, there are limited morphological characters that can be used to differentiate some of these taxa [8, 39]. Together these results imply that assigning separate generic status to some species within Somniosidae may be an overrepresentation of the true diversity within the family.

Occurrence and significance of bioluminescence in Squaliform sharks

The Bayesian inference estimated with BEAST [40, 41] is widely congruent with the maximum likelihood phylogeny (Fig. 1, Additional file 1: Figures S3 to S6).

Results from node time estimates based on 172 loci support a squaliform shark radiation beginning in the Lower Cretaceous and continuing through to the Upper Cretaceous (Table 1). A sister-group relationship of non-luminous Squalidae with a clade comprising all other Squaliformes is strongly supported as the most ancient split of extant Squaliformes (Table 1) and is consistent with the fossil record [25, 29]. Centrophoridae rise in the Lower Cretaceous, followed by the splitting of Dalatiidae, Somniosidae, Oxynotidae and Etmopteridae, which also aligns with the sequence of appearance of these taxa in the fossil record. However, 95 % confidence intervals are large, preventing exact estimates (Table 1). A second radiation occurred within Etmopteridae and Somniosidae in the Upper Cretaceous and the beginning of the Palaeocene (Table 1), again, a time period characterized by profound changes in the marine environment including the deep-sea. As discussed in [23], the Eocene recovery phase and the admixing of the deep-sea by the establishment of the circum-antarctic current at the beginning of the Oligocene, may have set the stage for this second radiation.

Table 1 Node time estimates for major splitting events Full size table

Novel ecological opportunities after oceanic anoxic events have been hypothesized to trigger adaptive radiation of sharks in deep-water environments in the Lower Cretaceous [10, 24]. Results from the MEDUSA [42, 43] analysis indicate a background diversification rate r = 0.02. An elevated diversification rate was detected for families Etmopteridae, Dalatiidae, Oxynotidae and Somniosidae, (r = 0.05) and the radiation of the species-rich genus Squalus (r = 0.15, Fig. 2).

Fig. 2 Chronogram resulting from the BEAST [38] analysis with estimated shift in the diversification rate. Background rate r = 0.02. The black stars indicate significant increase in the diversification rate to r = 0.15 (radiation Squalidae) and r = 0.05 (Etmopteridae, Oxynotidae and Somniosidae) estimated with MEDUSA [39, 40]. Scale bar in millions of years. Numbers at branches refer to node numbers given in Table 1. Pie charts indicate the probability that ancestral taxa are luminescent (blue) or not (red). Families Etmopteridae and Dalatiidae were coded as luminous as well as the genus Zameus within Somniosidae. * = Node calibrated with information from the fossil record (Table 2) Full size image

We reconstructed ancestral character states in order to test the hypothesis that bioluminescence evolved in conjunction with the diversification of the Dalatiidae, Etmopteridae, Oxynotidae and Somniosidae. In the first analysis, we coded Dalatiidae and Etmopteridae as luminescent. Results from this analysis indicated that the common ancestor of families Dalatiidae, Etmopteridae, Oxynotidae, and Somniosidae was already likely carrying luminous organs. Interestingly, Somniosidae have been widely accepted as non-luminous [2, 23, 30, 31, 44]. However, Shirai [8] suggested that all Somniosidae are luminescent except for the genus Somniosus, which may have secondarily lost the ability to produce light.

We reviewed the presence of photophores in Somniosidae and Oxynotidae, by inspecting the ventral surface area of several specimens housed in zoological collections. The inspection of skin samples from Zameus squamulosus revealed clear presence of epidermal photophores (mean diameter = 41.75 ± 1.95 μm, density = 26 units mm−2, PAP = 3.74 %) in this taxon (Fig. 3). The majority of these organs appeared to be ring-shaped and covered with translucent dermal denticles. Zameus photophores are visible as open dark circular plaques, typical of functional photophores that are capable of producing light. Indeed, this morphology is typically adopted by dalatiid and etmopterid photophores while glowing [44–48]; the translucent nature of Z. squamulosus scales would allow efficient transmission of underlying photophore light, similar to the observation of light transmission through the ventral scales of opisthoproctid fishes [49] or through the dorsal finspines of the velvet belly lanternshark, Etmopterus spinax [50].

Fig. 3 Microscopic photograph of an excised ventral skin patch of Zameus squamulosus (ZSM30966). Arrows indicate photophores in open state. Scale bar indicates 200 μm Full size image

Morphological data presented herein provide clear evidence that functional photophores are present within Somniosidae, at least within the genus Zameus (Fig. 3). All other inspected specimens showed no evidence of epidermal photophores. In light of this, the ancestral character state reconstruction was repeated incorporating results from the inspection of skin samples, i.e. coding the genus Zameus in addition to Etmopteridae and Dalatiidae as luminescent. Results from this analysis further increased the likelihood that the common ancestor of Dalatiidae, Etmopteridae and Somniosidae was luminescent (Fig. 2). The common ancestor of Centrophoridae, Etmopteridae, Dalatiidae, Oxynotidae, and Somniosidae is also implied to have been luminescent, but the likelihood is less compelling. A further analysis following [8] coding somniosid genera Centroselachus, Centroscymnus, Scymnodon, and Zameus as luminous further increases the likelihood so that the common ancestor of all Squaliformes except Squalidae may already have been luminescent (Additional file 1: Figure S8). This indicates that extant Centrophoridae may have secondarily lost their ability to emit light, i.e. that luminous organs may have already been present at the branching event giving rise to families Centrophoridae, Dalatiidae, Etmopteridae, Somniosidae, and Oxynotidae (Fig. 2). This suggests that luminescence evolved along and facilitated the Squaliform deep-sea radiation – a scenario that would be consistent with the elevated diversification rate detected for Etmopteridae, Somniosidae, and Oxynotidae. (Fig. 2, Additional file 1: Figures S8 and S9). We speculate that the common ancestor of families Dalatiidae, Etmopteridae, Oxynotidae, and Somniosidae was luminescent and used this to enhance camouflage by counterillumination as this is assumed to be the most basal function of shark bioluminescence [23, 28, 45, 47].

The occurrence of bioluminescence within the family Somniosidae is not surprising as especially the smaller sized genera (Centroselachus, Centroscymnus, Scymnodon, and Zameus) occur in sympatry with other luminous sharks such as etmopterids and dalatiids as well as a number of other luminescent deep-sea taxa including myctophid fishes which interestingly were estimated to have radiated in a similar time window [51]. Results presented here lend further support to the hypothesis that bioluminescence in sharks evolved only once [29, 47]. Work in progress will allow identifying all luminous taxa within the family Somniosidae.