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Advances in Taxonomy, Ecology, and Biogeography of Dirivultidae (Copepoda) Associated with Chemosynthetic Environments in the Deep Sea

Background Copepoda is one of the most prominent higher taxa with almost 80 described species at deep-sea hydrothermal vents. The unique copepod family Dirivultidae with currently 50 described species is the most species rich invertebrate family at hydrothermal vents. Methodology/Principal Findings We reviewed the literature of Dirivultidae and provide a complete key to species, and map geographical and habitat specific distribution. In addition we discuss the ecology and origin of this family. Conclusions/Significance Dirivultidae are only present at deep-sea hydrothermal vents and along the axial summit trough of midocean ridges, with the exception of Dirivultus dentaneus found associated with Lamellibrachia species at 1125 m depth off southern California. To our current knowledge Dirivultidae are unknown from shallow-water vents, seeps, whale falls, and wood falls. They are a prominent part of all communities at vents and in certain habitat types (like sulfide chimneys colonized by pompei worms) they are the most abundant animals. They are free-living on hard substrate, mostly found in aggregations of various foundation species (e.g. alvinellids, vestimentiferans, and bivalves). Most dirivultid species colonize more than one habitat type. Dirivultids have a world-wide distribution, but most genera and species are endemic to a single biogeographic region. Their origin is unclear yet, but immigration from other deep-sea chemosynthetic habitats (stepping stone hypothesis) or from the deep-sea sediments seems unlikely, since Dirivultidae are unknown from these environments. Dirivultidae is the most species rich family and thus can be considered the most successful taxon at deep-sea vents.


Introduction
Copepoda are estimated to contribute more than 15% to the total number of animal species known from deep-sea hydrothermal vents worldwide [1]. Almost 80 species are currently described from the orders Harpacticoida, Calanoida, Cyclopoida, Poecilostomatoida and Siphonostomatoida, but many more species await identification and description [2]. The Dirivultidae, a family belonging to the Siphonostomatoida, is the most diverse one of all animal families at vents with 13 genera including 50 described species. The most diverse genus is Stygiopontius with 21 species. Similar diverse faunal groups at deep-sea hydrothermal vents are Gastropoda with about 100 described species, including the prominent family Lepetodrilidae with 20 species and within this family the large genus Lepetodrilus (13 known species). Polychaeta are also represented with currently 111 species and the Polynoidae including 24 species [3].
In hard substrate ecosystems like many hydrothermal vents, copepods can be the most abundant and diverse meiofaunal taxon [4,5]. Copepods in general play an important role in various ecosystems, being usually the second dominant higher meiofauna taxon following the nematodes [6]. They are known from marine and freshwater plankton, marine sediments, cryptic habitats (soil, forest litter, terrestrial mosses, tree holes), subterranean habitats (springs, pools in caves), anchialine caves, deep-sea vents, and as animal and plant associates [7]. Their ecological importance is high and in some ecosystems as e.g. in the plankton, copepods are the main primary consumers. Copepods are essential for nutrient recycling and their fecal pellets are a central source for detritus feeders, but also the animals themselves are an abundant feeding source for macrofauna [7].
Dirivultidae are found in frequent and diverse numbers at hydrothermal vents around the globe. For this review we developed a simple identification table which should help scientists to identify these copepods easy in future. Ecological aspects such as abundance and diversity patterns are evaluated. We also provide an update on current distribution patterns of this unique family and discuss the origin of Dirivultidae.

Methods
We reviewed the literature of Dirivultidae, including all species descriptions and ecological studies. Original species descriptions were used to develop a key to genera and species. We investigated the occurrence of dirivultids in chemosynthetic habitats such as hydrothermal vents, cold seeps, wood falls and whale falls in the deep sea to provide a complete overview of the distribution of this unique family. In addition, we also considered trophic interactions and compared abundance and diversity patterns of Dirivultidae in various ecosystems and habitat types to gain insight into the ecology of these copepods. Biogeographical patterns were analyzed by separation into four large regions: the Atlantic, North East Pacific, East Pacific, and West Pacific, following the definition of Desbruyères et al. [3]. We use the thus obtained information to discuss the origin of the Dirivultidae.

Results and Discussion
Taxonomy Dirivultidae belong to the siphonostomatoid copepods and their morphological characteristics include: The body is cyclopiform with length ranging from 0.5 to 1.8 mm ( Figure 1A, Figure 2). The prosome is 4 segmented, the urosome 4-5 segmented in females and 5-6 segmented in males. The first urosomite bears the leg 5. The oral cone is short and robust formed by labrum and labium ( Figure 1D). In addition to the oral cone in the genera Ceuthoecetes, Dirivultus and Nilva a cutting borer is formed by the labium ( Figure 1E). Mandible, maxillule, maxilla, and maxilliped are present ( Figure 1A, 1D, 1E). Rami of legs 1 to 3 and exopod of leg 4 are 3-segmented ( Figure 1G). Endopod of leg 4 is 2-segmented (Figure 1 H). The development is as follows: females carry two eggsacks each containing one, frequently two (rarely more) large, yolky eggs; nauplii hatch as non-feeding lecithotrophic larvae, lacking mouth and labrum, and lacking a naupliar arthrite on the coxa of the antenna [8]. The exact number of naupliar stages is unknown; the lecithotrophic nauplius may moult directly into the first copepodid stage. Five copepodid stages with well developed mouth parts and gut follow, the sixth stage being the adult.
The presumably derived characters distinguishing Dirivultidae from Ecbathyriontidae and other siphonostomatoids are the 2segmented endopod of leg 4 (is 3-segmented in Ecbathyriontidae and many other siphonostomatoids) and the fusion of ancestral segments 3-8 in the proximal part of the antennule into one compound segment which is armed with 6 pairs of setae. Morphological observations suggest that Ecbathyriontidae, a family consisting of a single species (a new species and genus is in preparation, pers. com. VNI) (Ecbathyrion prolixicauda, Humes 1987) and found at hydrothermal vents, can be considered the only sister-group of Dirivultidae [9]. The synapomorphy of the taxon Ecbathyriontidae -Dirivultidae is the presence of a double segment in the female antennule. This double segment is armed with 2 pairs of setae and formed by fusion of two segments which correspond to the ancestral segments 15 and 16 [10].
The  Table 1). The genera can be mainly distinguished by the setation of the endopod of leg 4 ( Figure 1H). The genera Chasmatopontius and Fissuricola are considered as basal due to the existence of 3 inner setae on the distal (second) endopodal segment of leg 4. These 3 setae indicate that the ancestor had three endopodal segments on leg 4. The distal (third) and middle (second) segments of a 3segmented condition are fused into a distal double-segment in the 2-segmented condition. The presence of this former middle segment on the endopod of leg 4 is evidenced by the retention of 1 inner proximal seta of this segment (2 setae are indicated for the ancestor of siphonostomatoid). Ten other genera of dirivultids are characterized by a remarkably uniform 2-segmented endopod of leg 4. The distal endopodal segment of the 2-segmented endopod is armed with 2 setae at most, 1 terminal and 1 inner. The inner seta is lost in several genera of dirivultids. The proximal endopodal segment of leg 4 in dirivultids is armed with 1 inner seta at most as in the ancestral state of siphonostomatoids (this seta is lost in several genera of dirivultids). The endopod of leg 4 is lost completely in the monotypic genus Humesipontius. Table 1 is a key to genera featuring setation and some additional characters allowing genus identification. Schematic drawings of dirivultid morphology and important characters for identification are given in Figure 1.
Interestingly, only females or males are known in certain species despite the collection of sometimes thousands of specimens in a sample (see Table 5). For example, only females of Stygiopontius pectinatus, a species associated with the shrimp Rimicaris exoculata were found after inspection of more than 7400 individuals [20]. Whether the lack of finding both sexes has a biological background (e.g. parthenogenesis) or is simply due to wrong classification because of an acute sexual dimorphism remains to be studied, for example by life mating observations or by using genetic tools. Indeed, COI analyses of Stygiopontius hispidulus helped to find the male of that species (SG in prep.).

Ecology
Occurrence. Dirivultidae occur at deep-sea vents but have not been found in other chemosynthetic habitats such as shallow vents, seeps, whale falls (see Table 6) or wood falls nor in deep-sea or shallow-water sediments (PMA pers. obs.). At vents, however, they are not restricted to areas with vent flow, but can also survive away from vents on the bare basalt along the axial summit trough. Several species were encountered about 10 meters away from vents in the axial summit trough at the 9u509N East Pacific Rise (EPR) region [25]. Also Aphotopontius acanthinus and Stygiopontius hispidulus were recently detected in samples taken about 1 km offaxis in the 9u509N EPR region (SG pers. obs.).
Apparently, dirivultids are specialized to colonize hard substrate. Their relatively large body with powerful swimming/ crawling legs suggests that they are well adapted to an epibenthic life style [26], but they might not be able to live within vent and PLoS ONE | www.plosone.org seep sediments. However, while surfaces of tubeworms, mussels and other foundation species are colonized by dirivultids at vents, they are not inhabited by dirivultids at seeps. Further, whale bones and wood providing large surfaces for colonization are also devoid of dirivultids. We think that the large, continuous area of suitable substrate might play an important role for dirivultids to flourish at midocean ridges, but prevents them from colonizing relatively small patches of hard substrate of biotic origin (e.g. tubes, shells, bones, wood), which are surrounded by soft deep-sea sediments.
The occurrence of dirivultids is restricted to vents and the surrounding axial summit trough, which is in contrast to other meiofauna taxa. Harpacticoid copepod genera found at seeps and vents are usually unknown from deep-sea sediments, but their genera and sometimes even the species are known from shallow water sites (for more details see Martínez Arbizu et al. in prep.). Nematode genera detected at vents and seeps have been reported from deep-sea sediments but also from shallow regions (for more details see Vanreusel et al. in prep).
Most dirivultids are habitat generalists as they are able to live at different hydrothermal flux regimes and in different aggregates of megafauna. The majority of species was found in more than 2 different habitats, and only 38% of species were found in a single habitat (6 spp. at bivalves, 5 spp. at shrimps, 4 spp. at vestimentiferans, 1 sp. at alvinellids). 45% of species were detected in 2 habitats, most of them (11 from 19 spp.) in bivalve and in vestimentiferan habitats. 17% (7 spp.) were observed in three habitat types. Since it is known that those megafauna organisms  . The first step of genus identification is to analyze leg 4 endopod, afterwards the description of other characters should be followed. Other characters include number of segments and setation of other legs (terminology of different parts of legs see Figure 1A and 1G), number of setae on caudal rami and lobe presence/absence at anal somite (see for example Figure 1I showing Aphotopontius with 6 setae on caudal rami and anal somite without lobes), shape of antenna (A2) ( Figure 1B shows a typical antenna of Dirivultidae; Figure 1C shows the antenna with a hook like claw as typical for the genera Ceuthoecetes, Dirivultus, and Nilva), and number of segments in antennule (A1  First, species of this genus can be distinguished by the ratio of length to width of the caudal ramus (caudal ramus l:w). Second, species can be discriminated by the presence or absence of spiniform processes (S) on the genital somite of female (f) and male (m) (e.g. Figure 1I shows A. acanthinus with a caudal ramus ratio of length:width with 2:1; the female genital double somite has spiniform processes). Other species characters include the shape of various parts of the body (i.e. the maxilliped (mxp), see Figure 1F). doi:10.1371/journal.pone.0009801.t002 are found at distinct flux regimes (alvinellids and shrimp at high flow with temperatures .50uC, tubeworms at vigorous flow with moderate temperatures (,30uC), bivalves at low flow (,15uC) [31,32]), most dirivultids must be able to tolerate a wide range of hydrothermal fluid flux regimes. Information on where exactly and how dirivultids live is rare, since this often requires direct observations. Up to 10 copepods were counted per shrimp (Rimicaris exoculata) on the Mid-Atlantic Ridge. They were located on the mouthparts among dense bacteria growth, in the gill chambers, and/or probably were also swimming freely among shrimp swarms [20]. The close-up of a video camera from the submersible showed that dirivultids are crawling on alvinellid tubes colonizing sulfide chimneys at the East Pacific Rise (SG, MB pers. obs.). In this habitat type, temperatures among worms are ranging from 40uC to 100uC, sulfide concentrations can be above 1000 mM and oxygen is depleted [33,34]. Two of those dirivultid species, Benthoxynus spiculifer and Scotoecetes introrsus (both found in association with Paralvinella spp.), were investigated more in detail and exhibited high hemoglobin concentrations, with a very high and temperature sensitive oxygen affinity. This could be one of the crucial adaptations to live in lowoxygen environments [35,36].
Interestingly, there are often less males than females in dirivultid populations. For example, the female to male ratio at JFR was 7.6:1 for Stygiopontius quadrispinosus, 10.6:1 for Aphotopontius forcipatus, and 1.5:1 for Benthoxynus spiculifer [5]. Also, many species from tubeworm and mussel associated communities from the Northern EPR showed a female bias or even completely lacked males (Aphotopontius hydronauticus, A. probolus, A. acanthinus). But also, certain species such as Ceuthocetes acanthothrix, C. introversus, and Scotoecetes introrsus were male dominated [39].
Dirivultid copepod communities are less species rich at high flow alvinellid habitats than at low flow mussel and tubeworm habitats. Copepod communities associated with the alvinellid Paralvinella sulfincola at high temperature vents (communities sampled 4 cm away from 255uC peaks) at JFR were highly dominated by Stygiopontius quadrispinosus (80%), followed by Benthoxynus spiculifer (almost 20%) [5]. A similar dominance pattern was also found at high temperature vents of the EPR, where S. hispidulus was the most successful species in alvinellids Alvinella pompejana and A. caudata habitats [25]. In total 10 species are known from the alvinellid habitat (Table 5).
Trophic interactions. Most dirivultid species can be considered primary consumers and are grazing on bacterial mats and detritus [26,39]. This could be inferred by analyses of mouthparts and by the finding of partly dissolved bacteria and mucus in the foregut of specimens [26,38]. Copepods associated with shrimps were feeding on bacteria located on the shrimp mouthparts or on bacteria in the water column [20]. Detailed stable carbon and nitrogen isotopes in combination with fatty acid composition and morphological examination proved that Stygiopontius quadrispinosus and Benthoxynus spiculifer are mainly bacterivorous and, interestingly, food partitioning at the same trophic level occurred between these two species. S. quadrispinosus had a small mouth opening (,5 mm) and its diet was based on specific bacterial strains, composed of autotrophic bacteria. In contrast, B. spiculifer had a larger mouth opening (,20 mm) and was feeding on various autotrophic and heterotrophic bacteria, [61].
Only members of the genera Ceuthoecetes, Dirivultus, and Nilva have a different form of feeding, and are thought to feed on vestimentiferans [26]. The oral cone of these parasites is cylindrical and the labium is transformed into a cutting borer

Vent infauna
( Figure 1E). Photographs of vestimentiferans showed round wounds in the tentacular crown which were thought to be inflicted by Dirivultus dentaneus. However, it is also stated that indentations could be an artifact caused by the fixation [12]. Dirivultus spinigulatus was observed feeding on vestimentiferan plume filaments [21]. Dirivultids are a food source for macrofauna. Stable isotope studies on Paralvinella showed that copepods were part of its diet. It was hypothesized that copepods were consumed along with debris while the animal was grazing on the chimney surface [61]. It is unknown yet, but highly probable, that also many other macrofauna species feed on dirivultids.

Biogeography
Dirivultids are highly successful in their distribution since they are known from 4 main biogeographic regions, the Atlantic (A), North East Pacific (NEP), East Pacific (EP), and West Pacific (WP) (Figure 3; Table 5; [4,5,9,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][27][28][29][30]). A total of 13 genera with 50 species are currently known and most are endemic to a single region. Only five species occur in 2 regions and those belong to the two most diverse dirivultid genera Stygiopontius and Aphotopontius. We are not aware of any other region studied, in which dirivultids did occur. It has be taken into account that the majority of studies was historically carried out in the East Pacific. Therefore we expect that future collections will improve our knowledge of the distribution patterns in this family.
The diversity hotspot is the East Pacific with 33 species from 10 genera. Four genera with 4 species are known from the North East Pacific and 3 genera with 6 species from the West Pacific. In the Atlantic, a total of 3 genera with 12 species are currently recognized.
Nine of the 13 genera are endemic. Six genera are restricted to the East Pacific (Ceuthoecetes (4 Table 5).
The genus Stygiopontius has representatives in all four regions (EP: 11 spp.; A: 9 spp.; WP: 4 spp.; NEP: 1 sp.). Aphotopontius was found in the Atlantic (2 spp.), North East Pacific (1 sp.) and East Pacific (8 spp.). Benthoxynus is known with a single species each from the North East Pacific and the East Pacific, and Dirivultus from the West Pacific (1 sp.) and from off California (Dirivultus dentaneus; not at vents) ( Figure 3). However, only five species are known from 2 regions. The Atlantic and East Pacific share the species Stygiopontius mirus and S. rimivagus, the Atlantic and the West Pacific have S. pectinatus in common, the Atlantic and North East Pacific Aphotopontius forcipatus, and the East Pacific and the West Pacific S. stabilitus.
Dispersal of copepods in the pelagial is often but not exclusively during their copepodid stage [6]. Adults and copepodid stages of Rimipontius mediospinifer, Stygiopontius cladarus, S. pectinatus were found in plankton at 80-300 m above vents in the Mid-Atlantic Ridge Figure 3. Worldwide distribution of dirivultid genera. Current findings of dirivultid genera on mid-ocean ridges and back-arc basins in the Atlantic (red color code), North East Pacific (green color code), East Pacific (blue color code), and West Pacific (purple color code). The number of species is given between brackets. Map modified after Van Dover et al. [69]. doi:10.1371/journal.pone.0009801.g003 [28]. Other dirivultids from 9u509N at the EPR were caught in sediment traps positioned around and above vents (Lauren Mullineaux pers. com., SG pers. obs.). However, copepodids have also been sampled from tubeworm and mussel associations suggesting that at least part of the copepodid development is also possible within the benthos [39]. Although detailed studies on dispersal abilities (such as duration of nauplii and copepodids stages, their buoyancy and feeding strategies) lack, the first observations of copepods and their copepodids in the plankton give a hint that the global distribution of Dirivultidae may have been possible due to long-distance dispersal via ocean currents.

Origin and phylogenetic relationship
The distribution of extant dirivultids points to a pathway of immigration from shallow waters, and not from the deep-sea sediments, nor from other deep-sea chemosynthetic habitats as it has been suggested for many other vent animals [62,63]. Dirivultidae are only known from deep-sea hydrothermal vents and from the axial summit trough, but are unknown from deep-sea sediments. We conclude that other deep-sea chemosynthetic habitats did not facilitate immigration as stepping stones towards vents [64] or that dirivultids belong to the wide-spread sulphophilic fauna, because this family is unknown from seeps, whale falls, or any other reducing ecosystems. The only exception is the species Dirvultus dentaneus, which was once collected from the siboglinid tubeworm Lamellibrachia barhami at 1125 m depth off southern California [12]. L. barhami is known from the subduction zone cold seeps on the North America continental margin and from a sedimented hydrothermal region at Middle Valley on the Juan de Fuca Ridge [3]. Due to its limited distribution, it is also unlikely that dirivultids recently originated from a widespread fauna of generalists. Whether dirivultids have a long term in situ evolution remains to be tested. For small animals, immigration via their foundation species could be another option to invade the vent habitat. However, we suggest that alvinocarid shrimp, vestimentiferan tubeworms or bivalves did not act as ancestral carrier species. These megafauna species invaded the vent ecosystem via seeps, but dirivultids are not found there [63]. Alvinellid polychaetes are only found at vents, and the order Terebellida (to which alvinellids belong to) is found in shallow waters [63]. We propose that it is most likely that the dirivultid ancestor immigrated from the shallow water, the habitat where nowadays most Siphonostomatoida are found in association with various invertebrates and vertebrates [65]. Maybe, invasion was possible via the hard substrate ecosystem of mid-ocean ridges from shallow waters towards greater depths.
Dirivultidae are considered to have a basal position within the large order Siphonostomatoida due to the presence of an simple oral cone with a loosely associated labrum and labium, instead of a complex oral structure called siphon (with a fused labrum and labium) as found in many other siphonostomatoids [66]. The Siphonostomatoida includes more than 40 families with clear morphological distinction from other copepods (by the formation of an oral cone) but with unresolved phylogenetic relationships [67]. Siphonostomatoids live in association with other animals and most of them are animal parasites exhibiting a siphon for cutting and/or sucking. Two thirds of the species (with a total of .1550) are described as parasites of fishes and mammals, the other third are parasites or associates of invertebrates such as ascidians, polychaetes, bryozoans, cnidarians, crustaceans, echinoderms, or sponges [7]. In contrast, most dirivultids are not parasitic, but are free-living and bacterivorous and often live in aggregations of invertebrates at hydrothermal vents [26,61]. The bacterivorous feeding type (as seen from the simple mouth structure) of dirivultids suggests that they are basal to the other siphonostomatoids.
The phylogenetic relationships within Dirivultidae are unsolved yet, as detailed morphological comparisons and genetic analyses are by far not complete. The evolution of the formation of the oral cone (a key character of siphonostomatoids) has led to controversial ideas. The first idea, which in our opinion is the most probable one, is that the dirivultid ancestor had a simple oral cone (bacterivorous feeding). This is supported by the bacterivorous species Chasmatopontius and Fissuricola which are considered basal also due to the existence of 3 inner setae on the distal (second) endopodal segment of leg 4 (see Taxonomy). Over time, Dirivultidae adapted successfully to vents and developed there a more complex oral cone (evolution to a parasitic mode of life). In consequence, the ''cutting borer'', a modified distal disk of the oral cone formed by the labium of the parasitic genera Ceuthoecetes, Dirivultus, and Nilva would have evolved secondarily and independent from other parasitic Siphonostomatoida. The second idea is that the feeding apparatus in dirivultids could have evolved from a complex oral cone of secondary consumers (fused labrum and labium) back to a simple oral cone of primary consumers (with a loosely associated labrum and labium). The background of this hypothesis is that other families of the Siphonostomatoida are known to be mostly parasites, and in dirivultids, the antennae, maxillipeds and mandibles have the characteristic form known from those other parasitic Siphonostomatoida [26]. This would imply that Ceuthoecetes, Dirivultus, and Nilva are on the basis of Dirivultidae. However, it should be mentioned here that it remains to be clarified if these morphological features are related to adaptations of the feeding mode (parasitism) or to adaptations of the life style mode of dirivultids (which are free living on foundation species, so antennae could also be used to hold themselves on the foundation species and not to fall off). Interestingly, the Monstrilloida, a former copepod order that was recently placed within the Siphonostomatoida according to molecular analyses, are primary consumers. For this taxon, it has been suggested that they secondarily returned from an ectoparasitic to a free-living mode of life [68]. Only detailed morphological analyses in combination with gene analyses can help unravel the unsolved origin and phylogenetic relationships of Dirivultidae.

Future perspectives
Dirivultidae is the most diverse taxon at deep-sea hydrothermal vents. With the discovery of new vent sites and with the study of sites where macrofauna species are already known but not the meiofauna, species number is expected to increase further. Although they can be highly abundant in some vent habitats, only a few studies include this family in a broader ecological context. One goal is to take this family into account and the here provided key should help scientists to do so. Biogeographic patterns are expected to change with future collections; especially knowledge from the West Pacific region and the Indian Ocean is very scarce at the moment, and the polar regions remain completely unstudied. Origin and evolutionary processes are unclear yet, and in the future, genetic analyses will help to understand species distributions and speciation processes.