Genus level molecular phylogeny of Aegisthidae Gisbrecht, 1893 (Copepoda: Harpacticoida) reveals morphological adaptations to deep-sea and plagic habitats

Background The family Aegisthidae is known as typical component of deep-sea hyperbenthic waters that gradually colonized other marine environments. The phylogenetic relationships within this family have been examined here including hyperbenthic, planktonic, benthic forms and two associated Aegisthidae species. Results Ninety four specimens belong to 14 genera were studied using 18S and 28S rRNA and COI mtDNA. Bayesian analysis supports the monophyly of 10 genera whereas Andromastax, Jamstecia, Nudivorax and Aegisthus revealed to be paraphyletic. The first offshoot of the phylogenetic tree is a clade consists of the undescribed genus Aegisthidae gen.1 sister to the two monophyletic genera Cerviniella and Hase, whereas the other Cerviniinae members (represented by Cervinia and Expansicervinia) assemble a monophylum, sister to the hyperbenthic and planktonic aegisthid genera, resulting in the paraphyly of the subfamily Cerviniinae. Hence, we defined the new subfamily Cerviniellinae subfam. nov. encompassing the three benthic genera Cerviniella, Hase and Eucanuella. The subfamily Cerviniinae has been re-defined to include Cervinia, Expansicervinia and Paracerviniella. Members of the subfamily Pontostratiotinae were clustered into two clades, one consists of the genus Stratiopontotes sister to an undescribed genus + Cerviniopsis and Siphonis. The second contains Pontostratiotes sister to the members of the planktonic subfamily Aegisthinae, resulting in the paraphyly of the Pontostratiotinae. Therefore, the Pontostratiotinae has been re-defined to include only members of the genus Pontostratiotes; whereas the subfamily Cerviniopseinae has been re-erected and re-defined containing Stratiopontotes, Cerviniopsis, Siphonis, Aegisthidae gen. 2, Herdmaniopsis, Hemicervinia and Tonpostratiotes. Within this subfamily, the associated Siphonis clusters as sister to the Cerviniopsis represents an example of convergent evolution in which the possession of a stylet-like mandible and an oral cone reminiscent of the Siphonostomatoida. The planktonic Aegisthus, Andromastax, Jamstecia, Nudivorax and Scabrantenna confirm the monophylom Aegisthinae, sister to the Pontostratiotinae. Conclusions Our DNA based phylogeny reveals the deep-sea origin of Aegisthidae by placing benthic Aegisthidae gen.1 and Cerviniellinae subfam. nov. as the most basal lineages. Secondary adaptations to hyperbenthic and planktonic realms, as well as associated lifestyle were discovered here by the derived positions of Pontostratiotinae, Aegisthinae and Siphonis respectively.

In a morphology-based phylogenetic study conducted by Seifried and Schminke [22], the families Aegisthidae and Rometidae Seifried & Schminke, 2003(= Aegisthoidea Giesbrecht, 1892 were considered as the most basal lineages of Oligoarthra Lang, 1944; however the evolutionary history within the family Aegisthidae was not studied. Here, we attempt to resolve the phylogenetic relationships within this family based on a diverse collection of deep-sea aegisthid species inhabiting different habitats of the Atlantic and the Pacific oceans. Our molecular phylogeny of the Aegisthidae is based on small (18S) and large subunits (28S) of nuclear ribosomal RNA and the mitochondrial gene Cytochrome c oxidase subunit I (COI). Sixty species representing 14 genera of the three subfamilies were chosen for our phylogenetic analyses. This study represents the first attempt to reconstruct the evolutionary history of this diverse family using molecular methods and includes numerous taxa, some of them phylogenetically important, in all currently recognized subfamilies. A brief discussion of the main morphological adaptations is provided.

Results
Bayesian phylogenetic inference using two nuclear (18S and 28S rRNA) and one mitochondrial (COI mtDNA) gene sequences recovered Aegisthidae as a monophyletic clade. Ten monophyletic genera were recovered with high support values (posterior probabilities): Aegisthidae gen. 1 [21,22] only the Aegisthinae was recovered monophyletic, whereas, Cerviniinae and Pontostratiotinae were paraphyletic. The description of the new genus and species will be presented elsewhere. Figure 1 illustrates the phylogenetic tree of aegisthid species analyzed in this study in which five distinct clades have been revealed using Bayesian analysis:

Clade 1
Encloses the undescribed genus Aegisthidae gen. 1 sister to a monophylum containing the benthic Cerviniella and Hase. We proposed the new subfamily Cerviniellinae subfam. nov. enclosing the genera Cerviniella and Hase (included in the molecular analysis) as well as Eucanuella Scott T., 1901 (based on morphological apomorphies from previous studies). The undescribed genus (Aegisthidae gen. 1) has not been included in this subfamily following distinct morphological differences, therefore retained as a single independent lineage within the clade 1. Figures 2  and 3 show confocal scanning images from habitus and mouthparts of the undescribed genus Aegisthidae gen. 1 and representatives of the subfamily Cerviniellinae (Cerviniella and Hase) respectively.

Clade 2
Contains four genera Aegisthidae gen. 2, Cerviniopsis, Siphonis and the genus Stratiopontotes in which the last genus was recovered sister to all remaining genera within this clade. The subfamily Cerviniopseinae has been reerected here to contain these four genera. Also Hemicervinia Lang, 1935, Herdmaniopsis Brotskaya, 1963and Tonpostratiotes Itô, 1982 are recognized here as members of the re-erected subfamily Cerviniopseinae only based on morphological evidence. The associated genus Siphonis has shown to be a derived lineage of Cerviniopseinae sister to Cerviniopsis. Figure 4 presents scanned images from the genera Stratiopontotes and Siphonis.

Clade 3
A well-supported clade consists of two genera: Cervinia and Expansicervinia. Two distinct lineages were recovered within the genus Cervinia; one corresponds to the lineage previously known as Neocervinia Huys, Møbjerg & Kristensen, 1997(synonymized with Cervinia by [21]) sister to the rest of the Cervinia. However the both lineages are treated here as Cervinia due to a single specimen of the "Neocervinia-lineage" was available for this study. The re-validation of this taxon will be made elsewhere when enough material is available to support its taxonomic status. Here, the subfamily Cerviniinae is re-defined to enclose the genera Cervinia, Expansicervinia and Paracerviniella Brodsky, 1963 (the latest only based on morphological criteria). Figure 5 depicts the confocal scanning images form the representative of the subfamily Cerviniinae.

Clade 4
A highly supported monophyletic group including only members of the genus Pontostratiotes. Several lineages of Pontostratiotes were recovered which indicates the high diversity within the genus. Here we re-define the subfamily Pontostratiotinae as a monotypic taxon within Aegisthidae to accommodate species belonging to the genus Pontostratiotes only (Fig. 6d, e and f).
Two offbeat tree topologies have been resulted here based on two different character (nucleotide) partition settings implemented in Bayesian phylogenetic analysis from two different independent runs (see methods). The second tree topology (Additional file 1: Figure S1) contrasts with the first tree in 1) the position of Aegisthidae gen. 1, being sister to the genus Hase, 2) Cerviniella is paraphyletic, Hase and the Aegisthidae gen. 1 are nested within Cerviniella 2) the subfamilies Cerviniinae clustered as sister to an unstable clade containing Cerviniopseinae sister to Pontostratiotinae + Aegisthinae, 3) the genus Stratiopontotes is sister to a polytomy of Siphonis + Aegisthidae gen. 2 + Cerviniopsis, 4) the topology of the inter-clades within the genus Pontostratiotes is different in some lineages. This alternative phylogram displayed extremely low EES (estimated sampling size) value for the Log-likelihood estimation (LnL), it shows significantly low supports for some phylogenetically important clades and is incongruent with the principle of parsimony as accepting this topology would imply the character reversal of the unique modification of swimming legs present in Cerviniella (arrow in Fig. 3d) but also of the apomorphies of Cerviniellinae subfam. nov. as discussed below, to a morphologically unmodified condition in Aegisthidae gen 1. (Fig. 2b); hence this topology is presented in the supplementary material but not considered here for further discussion. The information about both MrBayes jobs including the alignments, nexus blocks, generated trees and the MrBayes log files (information about the runs) are available as Job1 and Job2 supplementary information (Additional files 2 and 3).

Discussions
The monophyly of Aegisthidae was examined here for the first time using molecular data of 18S, 28S rRNA and COI mtDNA. Our result confirms the monophyly of the family Aegisthidae established by Seifried [21] and Seifried and Schminke [22] based on morphological characters. The traditional morphological view that, consider three monophyletic subfamilies within Aegisthidae [21,22,29], is not supported in the present analyses of the family (Fig. 1). Our result agrees with the hypotheses of Seifried and Schminke [22] which proposed that, the genera Aegisthus, Andromastax, Jamstecia, Nudivorax and Scabrantenna Lee W. & Huys, 2000 represent an advance possibly monophyletic group within Aegisthidae. The morphological characters that allowed the inclusion of the Aegisthinae together with former Cerviniinae and former Pontostratiotinae (sensu [30]) in the family Aegisthidae are the unique form of the anal somite (elongate and tapering posteriorly) and the modification of the posterior seta 11 (terminology following [21]) of the maxilla into a strong large spine [21,22].
The unique spinous processes of the cephalothorax (arrow in Fig. 6b) and the extremely elongated furca (arrow in Fig. 6a) in Aegisthinae have been recognized by Seifried and Schminke [22] as derived characters shared with members of the former Pontostratiotinae (Pontostratiotes) (arrow in Fig. 6d,e) concluding that they belong to the same evolutionary lineage. However other harpacticoid reference literature (e.g. [29]) and even Seifried and Schminke [22] continued to consider former Pontostratiotinae and Aegisthinae to be valid, independent subfamilies. Our molecular analysis confirms the sister relationship between Aegisthinae and here re-defined Pontostratiotinae (which includes members of the Pontostratiotes only) and rejects the monophyly of the former Pontostratiotinae.  [24] In this study, the analyzed genera belonging to the Aegisthinae (Aegisthus, Andromastax, Nudivorax, and Jamstecia) have a complex topology ( Fig. 1) in which there is no evidence supporting the monophyly of none of the four genera. Conroy Dalton and Huys [27] described Andromastax as the second known genus in the Aegisthidae, arguing that the inclusion of the new species within Aegisthus (the only genus recognized at that time) was impossible without grossly extending its generic boundaries. Some of the morphological characters used to define Andromastax were based on a combination of strong plesiomorphic character states (e.g. Md palp 2-segmented and bisetose; Mx1 basis with 8 elements; A2 exopod with 3 elements; P5 exopod with inner setae; P6 with 3 setae) and clearly defined apomorphies (e.g. dorsal spinous processes on cephalothorax, cephalosome with lateral spinous processes near bases of A2; lateral processes on coxae P2-P4, ♀ second segment of A1 with 2 lateral processes on anterior margin, ♂ Mx2 allobasis and enp-1 with modified pinnate spines [27]. We suggest that the dorsal spinous processes on the trunk somites and A1 (arrow in Fig. 6b, e) represent a synapomorphic character for the lineage formed by Aegisthinae + Pontostratiotinae which were lost in some members of Pontostratiotes and in the genus Nudivorax. Lee and Huys [19] described three monotypic genera of Aegisthinae from hydrothermal vents and cold seeps in Japan: Nudivorax, Scabrantenna and Jamstecia. Nudivorax is characterized by a complete lack of integumental surface reticulation and lack of spinous processes on both cephalosome and body somites which was considered to be unique among the Aegisthinae. Scabrantenna (missing from this analysis) is distinguished from other genera because of its sexually dimorphic A1 and its prehensile A2 (presumably used for mate guarding). The most distinctive character of Jamstecia is an elongated A1, resulting from secondary elongation of segment 1 (Fig. 6a, b), differing from other Aegisthinae in which the second segment is the longest. Lee and Huys [19] argued that Nudivorax could represent the most primitive genus within the family because of the plesiomorphic state of female A1 (which retains the maximum number of setae expressed in the family) and male mouthparts (retaining the full complement of armature as found in females) suggesting an early divergence within the subfamily. The above mentioned authors also discussed that Scabrantenna represents a transitional genus between the primitive Nudivorax and Andromastax compared with the advanced Aegisthus and questioned the position of the genus Jamstecia due to the lack of material for comparison. Lack of support in the monophyly of these four genera in our study demands providing DNA sequences from the type species of these genera for deeper molecular and morphological analysis. The materials we have sequenced correspond morphologically to the genera assigned in Fig. 1 but the species are not the name bearing types.
Here we, re-validate and re-define the subfamily Cerviniopseinae as a well-supported monophyletic group that includes the genera Stratiopontotes, Siphonis, Cerviniopsis, Aegisthidae gen. 2., Hemicervinia, Herdmaniopsis and Tonpostratiotes. Stratiopontotes is here shown to be the most basal genus within the subfamily Cerviniopseinae. This genus mostly follows the ground pattern of the Aegisthidae in both cephalic and thoracic appendages, converging the molecular analysis with the morphology. Members of the genus Herdmaniopsis have been recognized as primitive forms within the former Pontostratiotinae by Lang [31], Brotskaya [9] and later by Ito [28]. The last author particularly emphasized the morphological similarities of the unarmed cephalic shield and thoracic pleuro-tergites of Stratiopontotes ( Fig. 4a) and Herdmaniopsis, suggesting the close relationship between those genera. Herdmaniopsis has no representative in our molecular study, however we agree with Ito [28] and believe that Herdmaniopsis may be sister to Stratiopontotes in a basal position within Cerviniopseinae. Additionally, we agree with the clear separation of both genera because of the absence of setae on the first endopodite of the maxilliped and the shortened of the A1 in Herdmaniopsis, among other characters. The only known associated genus of the family Aegisthidae, Siphonis, is shown here to be sister to Cerviniopsis. This genus is characterized by its siphonostomatoid-like mouthparts which include an elongated oral cone (formed by labrum and labium; that are sealed together by complex arrangement of overlapping ridges and grooves), a highly modified mandible (stylet-like) and maxillule and maxilla with the same shape as in siphonostomatoids (arrow in Fig. 4e,f) [21]. The short oral cone in Paninarthra families shows a similar morphology to the primitive families Asterocheridae Giesbrecht, 1899 and Dirivultidae Humes and Dojiri, 1980 of Siphonostomatoida [32,33]; whereas, an elongated oral cone and highly modified mandible in Siphonis is considered a homoplasy with some particular Asterocheridae species such as Acontiophorus scutatus (Brady and Robertson, 1873) and Scottocheres elongatus (Scott T. & Scott A., 1894) both associated with Porifera. These similar modifications are characteristic of those associated copepods and have the functional task of penetrating and feeding from pores and channels in sponges (for a detailed discussion of the morphology and mouthpart modifications in the genus Siphonis see [26]).
Aegisthidae gen. 1 is, genetically, a well-supported independent lineage, sister to the subfamily Cerviniellinae subfam. nov. This undescribed genus was excluded from the Cerviniellinae subfam. nov. based on the distinct morphological differences between this genus compare to Hase and Cerviniella. The taxonomic status of the undescribed genus needs to be studied in details when additional material become available. Furthermore this genus is morphologically similar to the genus Stratiopontotes in the general armature and segmentation of A1, A2, mandible, maxillula, maxilla, maxilliped and P1-P5 (Fig. 2b, c). However it can be easily distinguished from Stratiopontotes because of its divergent furca (arrow in Fig. 2b), the robust and slightly projected labrum (arrow in Fig. 2c), the ratio of P1 endopod-exopod and; its general shape -being more robust and moderately dorso-ventrally flattened than Stratiopontotes-among other characters. Morphologically, the undescribed genus Aegisthidae gen. 1 agrees with the ground pattern of Harpacticoida in several important characters; one of them is the incomplete fusion of the first pedigerous somite from the dorsal cephalic shield (arrow in Fig. 2a shows dorsal suture). The complete fusion of the above mentioned somites has been considered as synapomorphy for the Syngnatharthra group which includes all families within Harpacticoida except for Aegisthidae and Rometidae [22]. However a complete secondary separation of the cephalosome and first pedigerous somite is presented in some species of Syngnatharthra as Chappuisiidae Chappuis, 1940, Phyllognathopodidae Gurney, 1932 and Latiremidae Bozic, 1969; or partially retained in members of Rotundiclipeidae and Tachidiidae Sars, G. O. 1909.
The subfamily Cerviniellinae subfam. nov. is here redefined to enclose members of the genus Cerviniella, Hase and Eucanuella, the latest is not included in the molecular analysis. Eucanuella is included within this subfamily because of the fusion of the first pedigerous somite to the cephalosome (similar to Cerviniella; arrow in Fig. 3e), the short A1 bearing several elements transformed into spines (arrow in Fig. 3f), the presence of strong hook-like lateral projections on the genital double somite (arrow in Fig. 3e, f) and the P1 exp. bended inwards (similar to Hase; arrow in Fig. 3a), the last three characters considered here as apomorphies to the Cerviniellinae subfam. Nov.
The genera Neocervinia and Pseudocervinia were synonymized with Cervinia, based on a 'strict consensus tree' [21], who considered the female one segmented P5 (arrow in Fig. 5a) as the only apomorphic character of Neocervinia. Later, Park et al. [20] endorsed the generic status of Neocervinia adding the presence of an aesthetasc (incorrectly referred by the authors as sensilla) on the A1 second and third segments as another synapomorphy for the genus (see [19,34] for detailed characters). Our molecular analysis is incongruent with synonymizing Neocervinia with Cervinia, however re-validation of this taxon is postponed providing more material from this genus.

Conclusions
The family Aegisthidae has colonized a variety of habitats in the course of its diversification (Fig. 7). The deep- Fig. 7 The ecological adaptations of the Aegisthidae, showing the gradually colonized habitats by line drawings of the representative morphotype of each branch. The arrows show the evolutionary history considered for the Aegisthidae from its benthic ancestors sea benthic origin of Aegisthidae [9,35] is demonstrated by the basal lineages such as Aegisthidae gen. 1 and Cerviniellinae subfam. nov. These species live on and in the sediments, but may also swim in the water close to the bottom (epibenthic). Broad spade-like appendages which are associated with burrowing lifestyle [13,36,37] are adaptations in benthic Cerviniellinae subfam. nov. species. These adaptations involve a stout and robust body shape, commonly with short furca, reduction in the length of the A1 segments (always less than one fifth of the total length of cephalosome), the development of robust modified setae in the A1, reductions in swimming legs (e.g. P1 endopod reduced to a single segment or absent in some species), swimming legs bent inwardly and with strongly developed spines and processes that may help in sediment removal.
Secondary adaptations to a pelagic existence in Aegisthidae [25,38] are clearly distinguished in Pontostratiotinae in which extremely long furca, well developed spinous process and an increased body surface aid in avoidance of sinking are present. Members of the genus Pontostratiotes inhabit hyperbenthic water layers. The derived Aegisthinae, such as the planktonic species of the genus Aegisthus the increase in the length of the A1, A2 and furca and the segments of swimming legs are shown here to play an important role in the successful colonization of pelagic realm of the ocean [8,9,13,25,31,38]. The most advanced life style in this family is displayed by Siphonis, where mouth parts have been adapted to an associated life style, with a long siphonostomatoid-like oral cone, stylet-like mandible and siphostomatoid-like maxillula. Here, a comprehensive molecular phylogeny of the Aegisthidae shows that evolutionary trends in this family have begun from a benthic habit then to an epibenthic, hyperbenthic, planktonic then associated lifestyle.

Molecular analysis
DNA extractions were carried out using 40 μL Chelex (InstaGene Matrix, Bio − Rad) following the protocol of Estoup et al. [43] from whole individuals and supernatant was stored at − 20°C for later DNA analysis. Exoskeletons of the extracted specimens were fixed in glycerin on a glass slide and stored as a voucher for morphological identifications. DNA was analyzed for all 94 specimens available for this study (Table 1). Phylogenetic analyses were carried out using 914 bp of nuclear large (28S), 1792 bp of small (18S) subunits of rRNA and 741 bp of mitochondrial protein cytochrome c oxidase subunit I (COI). Amplification was performed using AccuStart II GelTrack PCR SuperMix (ThermoFisher Scientific) or Phire Green Hot Start II PCR Master Mix (ThermoFisher Scientific) in a 25 μL volume containing 9.5 μL H2O, 12.5 μL PCR Master Mix, 0.5 μL of each primer (10 pmol μL − 1 ) and 2 μL of DNA template. PCR products were checked by electrophoresis on a 1% agarose/TAE gel containing 1x GelRed. PCR products have been purified using EXO SAP (PCR cleaning, Thermo-Fisher Scientific) prior sequencing. Table 2 shows related PCR primers for each gene. Forward and reverse sequences were assembled and edited using Geneious (version 9.1.8 Biomatters; http://www.geneious.com). MAFFT v7.017 [50] was used to align trimmed DNA sequences for each gene under E-INS-i algorithm for nuclear genes and G-INS-i for COI [51]. The three alignments were concatenated and manually edited for ambiguous regions using Geneious v1.9.8. In order to root the tree, three species of Misophrioida Gurney, 1933 and two species of Canuelloida Khodami, Vaun MacArthur, Blanco-Bercial & Martinez Arbizu, 2017 [52] were chosen as outgroups. The phylogenetic analyses were conducted using Bayesian inferences using MrBayes MPI version v3.2.2 × 64 [53,54]. GTRGAMMA (General time reversible following gamma shape distribution) substitutional model were used for phylogenetic analyses as the best nucleotide fitting model for 18S and 28S rRNA calculated by jModeltest v0.1.1 [55] under Java. The codon model was implemented for the COI partition following the nexus block: lset Nucmodel = codon, code = metmt, omegavar = M3, nst = mix and rates = gamma. Two different MrBayes character partitions were provided and compared in two independent MrBayes jobs for the phylogenetic trees: job 1) considering 18S and 28S in one partition under nst = 6 (GTR) rate = gamma; job 2) allocating individual partition for 18S and 28S in which the nst = 6 and rate = gamma were applied to each partition separately. Posterior probabilities were estimated using 8,792, 000 generations through four simultaneous Markov Chains Monte Carlo for the number of 2 runs and sample frequency of every 1000 trees. The average standard deviation of split frequencies between 2 runs reached the value of 0.007 for job 1 and 0.004 for job 2. The ESS (estimated sampling size) is more than 300 for all estimated parameters in job1 in which the ESS value of the LnL has been estimated to 1408-1561 for both runs. However in Job 2 the ESS value of some parameters are less than minimum accepted (Supplementary information) which indicates that the posterior was not effectively sampled during this single run. A 50% majority rule consensus tree with median branch lengths was made, discarding the 25% of the first trees (relburnin = yes) from each job individually. All generated trees (.t files) and estimated priors (.p files) are available as supplementary information for both MrBayes jobs (Additional files 2 and 3). The gene fragments sequenced in this study are available in Genbank following the accession numbers MN536817 -MN536902 for 18S rRNA, MN536171 -MN536215 for COI mtDNA and MN535552 -MN535623 for 28S rRNA.
Remarks: the inclusion of the genus Paracerviniella within this family should be taken carefully, this taxon was described based only in a single male specimen. The variability and high dimorphism present in the Aegisthidae males makes difficult to decide the correct position of this taxon, the genus is temporally retained here until further specimens allowed a complete comparison.