The genus Gennadas (Benthesicymidae: Decapoda): morphology of copulatory characters, phylogeny and coevolution of genital structures

Species within Gennadas differ from each other largely only in male (petasma) and female (thelycum) copulatory characters, which were restudied in scanning electron microscopy and used as a basis for phylogenetic analyses. Twenty-six petasma characters and 49 thelycum characters were identified. All 16 recognized species of Gennadas and Aristaeomorpha foliacea (outgroup) were included as terminals. Four robust monophyletic clades were retrieved, described and diagnosed as new species groups. The thelycum characters had greater impact on tree topology and supported deeper nodes than did the petasma characters. We hypothesize that features of the thelycum evolved first followed by aspects of the petasma. Relatively more conservative characters include parts of the sternites of the thelycum and of the petasma, while the scuti and protuberances on the thelycum and the shape and subdivisions of the petasma lobes are evolutionarily plastic. We identified two groups of copulatory characters, which are likely coupled functionally and interlinked evolutionarily: (i) the external part of the petasma and the posterior part of the thelycum and (ii) the internal part of the petasma and anterior part of the thelycum. We reconstruct possible mating position during copulation for each of the new species groups presented here. We also present an updated key to genera of Benthesicymidae and key to species of Gennadas.

ALV, 0000-0002-6756-2468; AAL, 0000-0002-1105-8027 Species within Gennadas differ from each other largely only in male (petasma) and female (thelycum) copulatory characters, which were restudied in scanning electron microscopy and used as a basis for phylogenetic analyses. Twenty-six petasma characters and 49 thelycum characters were identified. All 16 recognized species of Gennadas and Aristaeomorpha foliacea (outgroup) were included as terminals. Four robust monophyletic clades were retrieved, described and diagnosed as new species groups. The thelycum characters had greater impact on tree topology and supported deeper nodes than did the petasma characters. We hypothesize that features of the thelycum evolved first followed by aspects of the petasma. Relatively more conservative characters include parts of the sternites of the thelycum and of the petasma, while the scuti and protuberances on the thelycum and the shape and subdivisions of the petasma lobes are evolutionarily plastic. We identified two groups of copulatory characters, which are likely coupled functionally and interlinked evolutionarily: (i) the external part of the petasma and the posterior part of the thelycum and (ii) the internal part of the petasma and anterior part of the thelycum. We reconstruct possible mating position during copulation for each of the new species groups presented here. We also present an updated key to genera of Benthesicymidae and key to species of Gennadas.
2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

Introduction
The family Benthesicymidae includes 40 valid species in five genera: Altelatipes, Bentheogennema, Benthesicymus, Benthonectes and Gennadas [1,2]. The family remains underexplored taxonomically, partly because the genus occurs in the deep sea. Indeed, four of five generic names start either with Benthe (from Greek benthos, 'depth of the sea') or Alte (altus, 'deep'). Owing to deep habitat preserved material is often in poor condition and many species are represented by a restricted number of individuals. Finally, Benthesicymidae are very similar in general morphology within the genera and only copulatory structures allow confident identification to species level. This paper is devoted to the genus Gennadas, the most abundant and diverse genus of Benthesicymidae, which encompasses 16 of the 40 species of the family (40%). Most species of Gennadas are abundant and widely distributed in all oceans and therefore were discovered and described already in the late nineteenth century or in the beginning of twentieth century. Only one species, associated with the seamounts of the distant Nazca and Sala-y-Gomez Ridges, was described later (Gennadas barbari:   [3]). Burkenroad [4], Tirmizi [5] and Kensley [6] described and figured a number of species of Gennadas, but made no attempt to revise the systematics of the genus on a global scale. Moreover, there are some ambiguities regarding some of the species in the most recent species list of the genus [2]. For example, Gennadas crassus Tirmizi, 1960 [5] is based only on a single female and no males have since been reported; Bentheogennema burkenroadi Krygier and Wasmer, 1975 [7] has, despite having greatly elaborate copulatory structures similar to those in Gennadas, until now not been placed in this genus.
In addition to the general biodiversity and taxonomic interest related to Gennadas, the genus offers a number of phylogenetic challenges due to the general similar morphology of many species. Indeed, regarding the external morphology, the genus is morphologically more uniform than the rest of the Benthesicymidae, which may be explained by the fact that they occupy very similar ecological niches in the marine habitats: all species are mesopelagic migrants occurring between 500 and 1500 m in the daytime and between 200 and 500 m at night in the Atlantic [8], Pacific [3] and Indian [9,10] Oceans.
However, genital structures (female thelyca and male petasmata) show an outstanding diversity in Gennadas and are therefore promising for phylogenetic reconstruction as was shown for other Dendrobranchiata [11][12][13][14]. Despite a greatly elaborate morphology, structural variation in the thelyca and petasmata between specimens are well known to be negligible, so these organs have traditionally been used as 'fingerprints' for species identification [3,5,10].
In this study, we provide an inventory of the global fauna of Gennadas. We also restudy and/or revise all available copulatory structures in search of suitable phylogenetic characters on which a new classification can be based. As in previous studies on the classification of other pelagic shrimps, we examine genital structures by the use of scanning electron microscopy (SEM). Based on the phylogeny, we test statistically the possibility of coevolution between male and female genital structures, test their contribution to the phylogeny and discuss questions such as: which genital structures arose first, female or male? Which genital structures are evolutionarily conservative and which are more plastic? Based on both phylogenetic and morphological considerations, we also propose hypotheses for how the complex female and male genital structures may operate and interact with each other during spermatophore deposition.

Material and morphological analysis
The material used for this study is primarily from Danish and Russian expeditions exploring the pelagic zone of the world oceans. Most material used for the study is stored in the crustacean collection of the Natural History Museum of Denmark (NHMD) (electronic supplementary material, appendix S1). The work involved sorting and identification of the about 200 samples of Gennadas; individuals were then selected for further study by SEM.
Prior to treatment for SEM, relevant parts (such as the thelycum, the petasma and the appendix masculina) of selected specimens were dissected in order to expose important structures for further study. The material was dehydrated in a graded ethanol series, critical point dried, mounted and coated with a mixture of platinum and palladium following standard procedures [15]

Phylogenetic analyses
Since the taxonomic validity of the genera of Benthesicymidae are uncertain and the phylogenetic relationships between them unknown, an outgroup was chosen outside the family. As molecular data have shown, Benthesicymidae is sister to Aristeidae [16] and we chose Aristaeomorpha foliacea (Risso, 1827) [17], type for genus by original designation, as the outgroup. All valid species of Gennadas and Bentheogennema were included as terminal taxa.
We used 49 thelycum-related and 26 petasma-related characters (electronic supplementary material, appendix S2). The data matrix is presented in electronic supplementary material, appendix S3. To estimate the contribution of the female and the male genital structures to the morphology-based phylogeny of Gennadas, we made three analyses: (i) one with only female characters included   characters included ('total evidence', Analysis 3). Data were handled and analysed under maximumparsimony settings using a combination of programs: WINCLADA/NONA, NDE (Nexus Data Editor) and TNT [18,19].
All characters were unordered (non-additive) and equally weighted. Trees were generated in TNT using the 'implicit enumeration' options. Relative stability of clades was assessed by standard bootstrapping (sample with replacement) with 10 000 pseudoreplicates and by Bremer support (algorithm tree bisection-reconnection, saving up to 10 000 trees up to eight steps longer). We considered the clades statistically significant if they were supported both by Bremer values greater than or equal to 3 and bootstrap values greater than or equal to 80.
In the resulting 'total evidence' phylogenetic tree, we considered each node as an evolutionary event and calculated how many character states within each group synchronously changed at each node of the tree. Changes in the character states at each node were considered as binary parameters (absent or present) and further analysed via ANOSIM and non-metric MDS analysis and hierarchical clustering (single linkage algorithm and Kulzcynsky similarity index).
Calculations and analyses were carried out with the use of Excel, STATISTICA and PAST v. 3.04 [20]. Correlations were considered significant if p < 0.05. G. elegans (figure 4a). The shape of each sternite is very characteristic in the various species and a number of distinct types could be recognized as seen in the following. Each of sternites may have a scutum of various size, shape, direction and armature. The scutumrelated characters are very characteristic for each species (figures 4 and 5). In some cases, scuti are greatly expanded and cover main part of the thelycum, either being posteriorly produced from S6 (figure 5a) or anteriorly produced from AS7 (figures 4b and 5h). In G. incertus (figure 5f ), a very unusual structure is produced from the central part of S8 and extending in all directions as a mushroom cap over S8.
In addition to scuti, species of Gennadas (all except G. elegans) have strong specialized setae of various size and shape. They may include medial setae on S6 (figure 4b,e,d), sublateral and lateral setae (figure 5c,d) on PS7.
The seminal receptacles in Gennadas are usually present (closed thelycum), although they may be developed to different extent. The female orifices may be guarded by scuti, which are either posteriorly extended from S6 (figures 4a,b,e,f and 5a,e) and/or anteriorly extended from AS7 (figures 4c,e,f,h and 5b). In four species, orifices are bordered by compound posterior and anterior projections of S6 and AS7, respectively (figure 5c,d,g,h). In G. tinayrei, the receptacles involve also fifth thoracic sternite (figure 4d), which have a long posteriorly directed spinose process.
The description of G. crassus Tirmizi, 1960 [5] was based on a single female individual and no records of either female or male have been made since. We have examined the holotype (focusing on the thelycum) and found no difference between this and the thelycum of G. gilchristi. Taking into account the very elaborate thelyca in all other Gennadas, which are used as a fingerprint for identification of species, we consider the former species a junior synonym of the latter.

Ultrastructure of the petasma
The petasma is consisting of PI, PM, PE and LA in all species except G. elegans, in which PM and LA are absent (figure 2a).
PI is covered with one to five rows of circinnuli along the medial edge. In all species except G. elegans (figure 2a), the distal part of PI is transformed into what we propose is a specialized grasping structure, which is characteristic for each species and may include protuberances and invaginations of various sizes and shapes (figures 2b-h and 3a-h). The grasping structure is well chitinized and rigid in most species, soft and folded in two species ( figure 3a,b). Sometimes, the grasping structure bears circinnuli greatly enlarged and/or transformed into hooks and/or spines (figures 2d,f and 3e,h).
LA is extending from that side of the petasma, which is directed towards the abdominal sternites. In some species, LA is very characteristic and has a greatly expanded apron-like apex (figure 2e,f ) or terminal hook (figure 3d). LA may be as a club, which is nearly orthogonal to the main lamella (    support (figure 6c). As in Analysis 1, the branching can be described as occurring at three levels: (i) basal branching (G. elegans and the core Gennadas), (ii) medial-level branching into four species groups and (iii) terminal branching with each terminal clade consisting of two species.

Synapomorphies
The core Gennadas (all species except G. elegans) was supported by synapomorphies mostly involving general morphological characters: presence of specialized setae, shape/division of thoracic sternites in the thelycum and presence/absence of lobes in the petasma (figure 6c). The medial-level branching was supported by synapomorphies mostly relating to the morphology of the medial part of the thelycum (AS7 and PS7); G. valens species group was additionally supported by characters relating to S8 and the petasma (figure 6c). The terminal clades (each consisting of two species) were supported by the characters related to detailed morphology: degree of extension and chitinization of sternites, presence and shape of specialized groups of setae, shape and development of scuti on the thelycum and lobi on the petasma (figure 6c).

Coevolution of morphological characters
ANOSIM multidimensional scaling and hierarchical clustering showed that morphology of different parts of copulatory structures have coevolved (figure 6d). The use of all available evolutionary events (nodes on phylogenetic tree-figure 6c) showed the following correlations: (1) between posterior part of the thelycum (S8) and external part of the petasma (PE) and (2) between anterior part of the thelycum (S6 and AS7) and interior part of the petasma (PI, PM and LA).
One part of the thelycum (PS7) was not grouped with any of the other copulatory structures.

Taxonomic implications 4.1.1. The status of Bentheogennema burkenroadi
Gennadas burkenroadi was described as Bentheogennema burkenroadi Krygier and Wasmer, 1975 [7]. The authors noted that this species has an intermediate position between Gennadas and Bentheogennema in having a single pair of lateral spines on the telson (as in Gennadas, Bentheogennema has two to four pairs) and podobranchs on the second maxilliped posteriad to the third pereopod (as in Bentheogennema, Gennadas has podobranchs on the second maxilliped only). The authors considered podobranchs as a 'primitive characteristic, a more important generic trait than the number of pairs of spines on the telson' [7, p. 49] and put the new species into Bentheogennema. We consider both traits above as equally important in the generic taxonomy of Decapoda and therefore refer to two additional evidences to resolve the position of B. burkenroadi. We examined type material of B. burkenroadi and found the thelycum and petasma of this species to be greatly elaborate and structurally similar to those seen in the core species of Gennadas (figures 3d and 5d), and significantly different from those in Bentheogennema. Our phylogenetic analysis showed that this species is deeply nested into the Gennadas tree and belongs to G. brevirostris species group. Hence, B. burkenroadi belongs to Gennadas which makes some changes in the diagnosis of Gennadas necessary: Gennadas Bate, 1881.
Emended diagnosis: Integument membranous; rostrum not reaching eye cornea, armed with one apical and one dorsal tooth, setose in between; carapace with distinct cervical and postcervical sulci reaching dorsal midline, antennal angle rounded, branchiostegal angle square, hepatic and branchiostegal carinae weak; abdomen with first to fifth somites dorsally rounded and sixth somite dorsally carinate; telson posteriorly truncate, with a single pair of movable posterolateral spines. Eyestalks with long tubercle; first maxilliped with exopod not segmented distally; fourth and fifth pereopods with dactyl slender, entire. Petasma as wide lamina with developed elongate PE; appendix masculina bilamellate, with inner lobe spinose; thelycum elaborated, formed by sixth to eighth thoracic segments.

New classification of Gennadas and new species groups
Burkenroad [4] found that in 'all species of Gennadas with independent spermathecal orifices (e.g., open thelycum) the distolateral lobe of the petasma is entire, not subdivided; whereas in all species with orifices contained within a common atrium (e.g., closed thelycum), the distolateral lobe is bifurcated'. On the basis of these findings, he divided Gennadas into two groups, which were followed hereafter. However, this subdivision of Gennadas could not be further substantiated by our work. First of all, the information revealed by SEM showed that in most Gennadas, the thelycum is more or less closed and the orifices are covered by the shields either anteriorly or posteriorly. Open thelyca were found only in G. incertus and G. propinquus (figure 5f,g), in which the PE (the distolateral lobe) of the petasma was either divided (G. incertus, figure 3f ) or entire (G. propinquus, 2G). Second, there are numerous exceptions from Burkenroad's rule, for example G. barbari (not known to Burkenroad [4]), and G. incertus both have shallow seminal receptacles and greatly elaborate petasma; conversely, G. elegans, G. kempi, G. capensis, G. parvus and G. sordidus all have deeply closed seminal receptacles and relatively simple petasma. Instead of Burkenroad's scheme, we propose a new phylogeny-based classification.
Gennadas consists of two major phylogenetic lineages: G. elegans and the rest of Gennadas. Contrary to the core Gennadas but like other Benthesicymidae (Bentheogennema and Benthesicymus), G. elegans has a simple petasma without LA, PM and grasping structure on PI. However, the morphology of the thelycum in G. elegans is elaborate and more similar to the core Gennadas than to the other genera. Our phylogenetic analysis did not support grouping this species with the core genus. Gennadas elegans probably should be placed in a separate genus Amalopenaeus (first description: Amalopenaeus elegans Smith, 1882 [21]) in order to keep the monophyly of Gennadas, but we are reluctant to do this until a global phylogenetic revision of the whole family Benthesicymidae is completed.
Phylogenetic analysis has shown that the core Gennadas further branches into four very robust clades, each consisting of two to five species. To better represent the phylogenetic information in the classification, we subdivide Gennadas into four species groups and treat G. elegans as separate from these.

G. elegans
Diagnosis: (figures 2a and 4a) Thelycum: specialized setae absent; sixth sternite with posteriorly directed scutum in the anterior part and with W-shaped posterior prominence; seventh sternite undivided, as a simple oval plate.
Petasma: PI without distal grasping structure, LA and PM absent, PE leaf-like. Both lobes of appendix masculina spinose.

G. valens species group
Diagnosis: (figures 2g,h and 4g,h) Thelycum: specialized setae present; sixth sternite simple subtriangular, without scutum; seventh sternite divided into two shields, anterior shield bat-like, lateral edges not reaching coxae, posterior shield trapezoid, with anterior incision, lateral edges not reaching coxae, anteriorly armed with a row of strong spines; eighth sternite with median groove in the posterior part, without scutum.
Petasma: PI with distal grasping structure, LA long and distally extended, directed nearly orthogonal to main lamina, PM apically cleft into smaller interior and larger exterior lobules, PE with rounded apical notch as bit of key. Outer lobe of appendix masculina unarmed.

G. brevirostris species group
Diagnosis: (figures 3c,d,g,h and 5c,d,g,h) Thelycum: specialized setae present; sixth sternite simple subtriangular; seventh sternite divided into two shields, posterior shield as long chitinized strip, laterally produced beyond coxae, with two groups of lateral setae in addition to two groups of sublateral setae. Species included: with five species: G. bouvieri, G. capensis, G. incertus and G. kempi.
Petasma: PI with distal grasping structure, LA, PM and PE present. Outer lobe of appendix masculina unarmed.

Key to genera of Benthesicymidae and key species of Gennadas
Key to Genera of Benthesicymidae

Phylogenetic implications
The phylogenetic tree shows that the medium-level clades (the species groups) are primarily supported by thelycum-related characters, while petasma-related characters are more important for the support of the terminal clades. The greater importance of the thelycum for the main branches of the tree is also illustrated when comparing the thelycum-based and the petasma-based MP trees: the female tree is much more resolved than the male tree and has the same topology as the 'total evidence' tree. It is remarkable that the terminal clades (species pairs) are similar in all trees. However, the clade support is weaker in the male tree, where some species group-level clades fail to appear. We here hypothesize that the thelycumbased characters were first to appear in evolution followed by the petasma-based characters. Among these possibly co-evolving characters are the structure of the sixth and seventh thoracic sternites and the presence of specialized groups of setae on thelycum.
Other thelycum-related characters are much less conservative. Even such remarkable speciesdiagnostic characters as scuti, occurring in different parts of the thelycum (from S6 to S8) and being very characteristic in shape, size and armature, seem evolved later in evolution, at the species level. In most species, they are not fully homologous, which is indicated by the fact that they occur on different somites.
The petasma is generally an evolutionary more plastic character than the thelycum. Only presence of the major lobes (LA, PM and the grasping structure of PI) determining the rough topology of the petasma is conservative. Such characters as further division and shape of the lobes, the presence of additional lobules and their serration, etc. are species-specific and have appeared late.
We thus suggest a 'lock and key' hypothesis and tentatively conclude that females of Gennadas were the first to evolve elaborate sexual structures externally (topology of sternites) later followed by males, in which a very specific petasma is present in each species. The lobules of the petasma, which are very specific in size and shape, probably evolved to fit species-specific parts of the thelycum (scuti and microrelief).

Coevolution of characters and functional morphology of copulation
An analysis of the coevolution between central aspects of the sexual structures showed a couple of statistically significant correlations: (i) between the external part of the petasma and the posterior part of the thelycum and (ii) between the internal and medial parts of the petasma and the anterior part of the thelycum.
The explanation for the coevolution between certain sexual structures in females and males must be that these structures interact functionally during copulation. However, in situ observations of copulation in Dendrobranchiata are very rare for coastal species and entirely absent for pelagic ones. Bauer [22] was among the first to observe and analyse copulation of the penaeid shrimp Sicyonia dorsalis. He found that the position of the male was at right angles below the female during copulations and that males were able to inseminate only the spermatheca on one side per successful copulation. He proposed that the petasma is not a sperm injection device; instead, this organ is used to hook onto the female thelycum, adjusting the proper position during copulation.
Assuming that a comparable mechanism exists in pelagic Gennadas, we reconstruct the details of spermatophore deposition based on which parts of the petasma and thelycum fit to each other. Figure 7 shows a hypothesized fit between male and female copulatory structures during copulation for four species representing each of the four species groups of the core Gennadas. It is clear that in all cases, PE of the petasma always fits to S8 of the thelycum, and that the grasping structure of PI of the petasma fits to the seminal receptacles of the thelycum. The grasping structure, which is synapomorphic for the core Gennadas, may serve for carrying the spermatophore and fixing it to the receptacles. It is noteworthy that the nearly terminal position of the grasping structure on the petasma facilitates receiving the spermatophores from the genital apertures, which face the dorsal (anterior) side of the petasma.
In some species, the petasma and the thelycum both have very peculiar structures, which provide further evidence for how female and male structures fit with each other during copulation. For example, G. incertus (figure 7a) has an unusual fork-like PE of the petasma, which may be passed under the cap of the mushroom-like structure on S8 of thelycum (which is also a unique structure). The apron-like apex of LA in the petasma of G. parvus is suitable for hanging onto the coxa of the third pereopod (figure 7b). The mitten-like PM of the petasma of G. scutatus and G. talismani may be adapted to pass under the scutum of the former and anterior setose projections of S8 of the latter (figure 7c,d).

Ecological and biogeographical implications
Recent analyses have suggested that the elaborate male petasmata are important for successful colonization of the pelagic zone by shrimp-like eucarids [23]. In the turbulent and hydrographically dynamic pelagic zone, successful copulation depends on perfect fixation and possible stimulation of mates during spermatophore transfer and thus on the presence of suitable copulatory structures.  In taxa such as Sergestidae, Luciferidae and Euphausiidae, the petasma is very elaborate consisting of branches with numerous lobi and processi, probably among the most elaborate in the animal kingdom. Surprisingly, the thelyca in the same families are simple and not greatly specialized. By contrast, the core Gennadas of Benthesicymidae is characterized by a simpler (but still elaborate) petasma and by the most elaborate thelycum among the eucarids. The thelyca of Gennadas species play a larger role in stable copulation and sperm transfer than in other eucarids.
Gennadas is among the most widely distributed pelagic genera. Indeed, four species (25%) are panoceanic: G. bouvieri, G. capensis, G. scutatus and G. tinayrei. Four species live in the Atlantic (G. brevirostris, G. elegans, G. talismani and G. valens), four species live in the Indo-Pacific (G. incertus, G. parvus, G. propinquus and G. sordidus) and two species live in the Indo-West Pacific (G. gilchristi and G. kempi). There are only two species with regional distribution: G. burkenroadi from the northeast Pacific and G. barbari from the southeast Pacific. The latter is benthopelagic and associated with seamounts of the Nazca and Sala-y-Gomez Ridges, which may explain its restricted distribution [3,10].
The greatly elaborate copulatory structures of Gennadas and the absence of significant individual variations in these structures both favour sexual isolation between species, which are otherwise ecologically similar in the mesopelagic habitat.