No specific permits were required for collecting the Trechini species of the genera Trechus and Geotrechus used for this study in France, because these genera are not protected. Licenses for collecting the four species of Aphaenops protected in France were obtained from the Directions Régionales de l’Environnement of the Midi-Pyrénées (A. crypticola, A. leschenaulti) and Aquitaine (A. loubensi, A. ochsi) (arrêtés N°2003–06, 25/2003, 2004–01). None of the species sampled are included in international lists for the conservation of endangered species of wild fauna (IUCN Red List of Threatened Species, Annexes of the Habitat Directive 92/43/EEC, Bern Convention or CITES).

One of the major challenges with subterranean beetles is to keep them alive from the cave to laboratory. Specimens were collected by hand, directly placed in a plastic container with humid moss and brought to the laboratory where they were anesthetized with ethyl acetate. We used four species of Aphaenops of the subgenera Aphaenops s. str. and Cerbaphaenops. We also studied one species of Geotrechus (G. gallicus) and one species of Trechus (T. bordei). We focused on the western clade of Aphaenops, with three representatives belonging to three distinct subclades: A. leschenaulti Bonvouloir, 1861, A. loubensi Jeannel, 1953, and A. ochsi cabidochei Coiffait, 1959 [25].

Anesthetized females were dissected in a saline solution (0.9%). After removal of the dorsal abdominal side and the intestine, the genital tract and the ovaries can be observed in place if they are not embedded in too much body fat. When the body fat was abundant, it was removed manually. Specimens were placed in a cavity slide and illustrated both before and after immersion in 70% ethanol to clarify some details inside the ovaries, such as the presence or absence of trophocytes and oocytes. All dissected parts were returned to alcohol for preservation.

Dry specimens were used for the examination of the ectodermal genital tract restricted to the cuticular intima, as many details are indiscernible on living specimens. Female abdomens were cleared in a 10% solution of potassium hydroxide (KOH) at room temperature for one to two days, then rinsed in water, placed in glycerol and dissected. The genital tract was then stained with Chlorazol Black E, placed in a cavity slide containing glycerol, and examined and drawn using a light microscope with a camera lucida. Pieces of specimens were stored in glycerol after examination. We follow the terminology of Deuve [29] for the female ectodermal genitalia.

The phylogeny was constructed using the following DNA fragments from selected species: two mitochondrial (5’ end of cytochrome c oxidase subunit 1 (cox1) and 5’ end of large ribosomal unit plus the Leucine transfer plus the 3’ end of NADH dehydrogenase subunit 1 (rrnL+trnL+nad1)) and two nuclear (small (SSU) and large (LSU) ribosomal units). Sequence data for all loci were generated in [24–25, 30] and available on GenBank (S1 Table). We aligned the sequences using the MAFFT online v.6 with the Q-INS-i algorithm and default parameters [31]. Maximum likelihood analyses were conducted on a combined data matrix with RAxML GUI [32–33], using three partitions corresponding to the cox1, rrnL+trnL+nad1 and nuclear fragments, with a GTR+I+G evolutionary model. We used the default values for other parameters of the search [32]. Character states were mapped to the tips of the phylogeny.

Trechus bordei possesses 5 to 7 polytrophic ovarioles per ovary (Fig 1A). The polytrophic type of ovariole is the rule within the entire group Adephaga. In polytrophic ovarioles, each follicle contains a number of trophocytes (nurse cells) together with one oocyte [35–37]. Ovarioles are tubular and elongate, attached together at the anterior end by their terminal filament. Germarium and vitellarium are distinct and the latter contains one or two small, immature basal oocytes. Pedicels are also elongated and seem to open directly into the median oviduct, the lateral oviducts being either extremely reduced or absent. The totality of the genital efferent system is enveloped in a thick layer of musculature (mainly circular muscle fibers, as illustrated in Fig 1E) to such an extent that the median oviduct, the vagina and the bursa copulatrix cannot be distinguished from each other. Between the ovaries there is a prominent median blind tube might be erroneously interpreted as a diverticulum of the genital efferent system, or a spermatheca or an accessory gland. However, treatment with KOH revealed that this tube is in fact the apical part of the bursa copulatrix tightly coated by a muscular sheath (see below). In one female specimen this tube-like projection was indistinct.

The ovariole number of Geotrechus gallicus was not clear and needs confirmation. Only a single female specimen was dissected and the ovaries were partly destroyed during the process. Nevertheless, in each ovary we were able to distinguish one well-developed ovariole plus several (2–3?) very thin ovariole-like structures in addition to the well-developed one. All the ovarioles were attached at the anterior tip by their terminal filament. The two well-developed ovarioles contained one or two small growing oocytes in the vitellarium. The thin ovariole-like structures appeared to be almost empty of cells. In Geotrechus orpheus, Deleurance-Glaçon [19] illustrated somewhat similar ovaries and counted two ovarioles per ovary.

Both species possess paired defense glands (pygidial glands) typical of Carabidae and the entire Adephaga [29, 38]. They open postero-laterally through the membrane between the mediotergites VIII-IX. The reservoirs are large; the acinous glands are deeply placed inside the abdomen and lie laterally on each side of the ovaries. They are connected to the reservoir by a remarkably long and convoluted collecting canal (Fig 1A).

Aphaenops loubensi (Fig 1B), A. ochsi (Fig 1C), A. leschenaulti (Fig 1D) and A. crypticola (Fig 1E) possess only a single polytrophic ovariole per ovary. These single-ovariole ovaries are very small, somewhat sac-shaped, and usually embedded within a thick layer of fat body; the terminal filament is indistinct. The germarium and vitellarium are not distinct from each other, the pedicel is almost non-existent and lateral oviducts are probably absent. The other parts of the efferent genital duct—median oviduct, bursa copulatrix and vagina—are entirely enveloped in a dense, common sheath of musculature, such that they cannot be distinguished from one another. However, in A. crypticola (Fig 1E) a tube-like protrusion and a small pigmented vesicle project out of the thick muscle sheath; these structures were interpreted as forming the spermatheca (see below). One female of A. crypticola was gravid (Fig 2); the dextral ovariole contained a single, conspicuous, mature egg, with numerous, large, lipid-like spherules. As shown in Fig 2, the 2.1 mm long egg occupied most of the abdominal cavity, representing nearly half the length of the female (4.5mm). In contrast, the left ovariole appeared minute, containing only a small basal oocyte. The two ovarioles may function alternately.

The paired defense glands are present in the four Aphaenops species examined. As in Trechus bordei, the acinous glands are located at the level of the ovariole and the reservoirs do not show any sign of reduction.

Examination of the female ectodermal genital tract after KOH treatment revealed several details through the cuticular intima lining. The female genital ducts of the five species examined are illustrated in Fig 3. A full description falls out of the scope of this paper and we only treat their general characteristics here, which have already been observed by Deuve [29] in other Trechini. There is a great diversity in the architecture of the genital ducts, even within the same genus (Fig 3B–3E). The following features are noticeable: i) the common oviduct, located ventrally as in other Adephaga, which is either long and straight or curiously collapsed upon itself (Fig 3B), as it is the rule in many insects, its intima bears internal minute spines; ii) the bursa copulatrix is present (Fig 3A, 3C and 3D) or absent (Fig 3B and 3E); iii), the spermatheca is lost, except in A. crypticola (Fig 3E); iv), the vagina is wide, being either simple (Fig 3A, 3D and 3E) or equipped with a posteriorly rounded diverticulum (Fig 3B) or with a large dorsal pouch (Fig 3C). Furthermore, in A. crypticola alone, we have observed numerous cuticular ductules scattered on the vaginal intima close to the opening of the spermatheca (Fig 3E), these being the efferent ductules of the secretory units of class III according to the classification of Noirot and Quennedey [39]. Except for these scattered secretory units, no glands are associated with the genital ducts.

In Coleoptera, the number of ovarioles per ovary can vary from one to 200 [40], and even up to 1000 in the genus Meloe (Meloidae) [37]. As stated by Robertson [40], during the diversification of Coleoptera the ovariole number has either been multiplied or reduced within groups, whereas in others, such as in Curculionidae, it has remained highly constant. In Carabidae, as for all Adephaga, ovariole number is still poorly documented, but it appears to be highly variable. Each ovary may contain from 4 to 40 ovarioles in Adephaga and from 4 to 18 in Carabidae [40]. In the Trechinae, although only known for a few species, the number of ovarioles ranges from 1 to 7 [19] (see Table 1).

In several groups, it is clear that the reduction of the ovariole number has been influenced by the life history of the species. For example, a reduction in the number of ovarioles is correlated with nesting behavior in Hymenoptera [41] and Scarabaeoidea [42]. The lowest number of ovarioles in Coleoptera is 1 or 0 in dung beetles (Scarabeidae s. str.) and this single ovariole is the left unpaired ovary typical of the family [43–47]. In this group, the extreme reduction of the ovariole number (1–0) is clearly related to the complex nesting behavior, with the digging of a nest and provisioning for each egg, so that the larva has all the food required for its development [42,46]. The great investment of the female—with the cooperation of the male in several species—does not allow for high fecundity and thus reduces the number of eggs laid. The loss of an ovary is exceptional in insects. Within Coleoptera it has been reported in Bledius spp. (Staphylinidae), a subsocial genus living in intertidal areas [48]. In this genus, females exhibit parental care [48–50]. However, in this case the remaining ovary has 6 ovarioles, the typical number for the family. Drastic ovarian regression to 1–0 ovarioles, as in dung beetles, is also observed in the holocyclic aphid species with sexual generation, particularly in the Phylloxeridae and Adelgidae, in which female sexuales, after mating, deposit a single egg (the “overwintering egg”) and then die (see [51–54]). Moreover, within a given aphid species, the number of ovarioles is highly variable between generations [55].

In social insects (Hymenoptera) ovariole reduction occurs in the workers, which possess very few ovarioles compared to the queen. In Cremastogaster (Formicidae), each ovary is reduced to one ovariole [56]. In Apis mellifera ovarioles vary from 1 to 30 [57–58].

Except for Drosophila (see [2]), very little is known about the ontogenetic process underlying ovariole differentiation. As stated by Green [2], the larval stages represent a critical period during which ovariole number is determined. In Scarabaeidae (Coleoptera), the reduction in ovariole number takes place during postembryonic development, after the formation of two symmetric gonads in which six ovarioles are differentiated, as in most Scarabaeoidea [59]. All the ovarioles, save one in the left ovary, are apparently sterile, remaining atrophied during organogenesis and finally disappear [59]. In several aberrant cases (females belonging to Scarabaeus, Onthophagus and Canthon) the persistence of a second either functional or non-functional ovariole in the left ovary has been observed [44, 59–61]. These cases show that the sequence of events may have broken down during organogenesis.

Various factors affecting the natural history of hypogean animals (scarcity of food in subterranean ecosystems, stable conditions and flightlessness) lead to a reduction in ovariole numbers. This may reflect the evolution of an extreme K-strategy in a harsh and stable environment [11]. The adaptation of animals to a subterranean life leads to remarkable morphological and physiological convergences [6,8,10,62–63]. In Aphaenops, it is likely that the ovarian regression is genetically determined and not environmental. This ovarian reduction is strongly correlated with both the very large eggs produced and the contraction in number of larval stages. Aphaenops lay eggs that are remarkably large in comparison with the body size of the female (e.g., ca. half of the body length of the female in A. crypticola, Fig 2). The reduction in ovariole number is very often associated with the large size of mature eggs in insects (e.g., [41,56,64]). The most striking example is the single “overwintering egg” laid by the female aphids during the sexual generation of Phylloxeridae and Adelgidae. In the woolly apple aphid (Eriosoma lanigerum Hausmann), the mature egg occupies the whole of the abdominal and thoracic cavity (see Figure 254 in [52]). In Aphaenops crypticola, the mature egg fills almost all the abdominal cavity. The huge eggs of Aphaenops spp. might be related to the contraction of the larval phase and might supply the non-feeding larva with the elements needed to complete its development. These elements are the proteid yolk (vitellins), which represents the major component of insect eggs, along with lipids and carbohydrates, which are present in considerable amounts [64]. Food supply is known to have a direct impact on the number of eggs laid in Carabidae [65]. The scarcity of food in caves may also impact the strategy of a reduction in the number of eggs laid. In beetles of the family Silphidae, flightlessness and food habit change are also known to be associated with a decrease in ovariole number and an increase in egg size [66]. The correlation between a reduction in ovariole number and subterranean specialization was first noted in Canarian cockroaches of the genus Loboptera (Dictyoptera, Blatellidae) [67]. Within Coleoptera, Deleurance-Glaçon noticed that although the correlation between troglobiomorphism, a shortening of the larval phase and a reduction in ovariole number is apparent within Trechini, this is not the case for Leptodirini (Leiodidae). In the latter group, the specialization to subterranean life is associated with a reduction in the number of larval instars, but this adaptation appears not to be correlated with a reduction of the ovariole number [19].

All the species of Aphaenops studied to date share the state of an ovary with a single ovariole. The genus Aphaenops is not monophyletic, and is represented by two independent subterranean radiations in the Pyrenees, one in the Eastern part (Cerbaphaenops), one in the Western part of the range (Aphaenops s. str.) [25]. Hydraphaenops, which is not monophyletic, was recently considered as a subgenus of Aphaenops, together with a new subgenus, Simaphaenops [26]. Most of the species belonging to these two groups are known by very few specimens, and the only species for which the number of ovarioles is known is A. (Hydraphaenops) ehlersi [15,19]. It has two ovarioles per ovary and an active larva ([19]; Table 1). Because A. (H.) ehlersi is phylogenetically closer to Cerbaphaenops than to the western Aphaenops s. str., the presence of more than one ovariole per ovary in this species indicates an independent reduction within the Pyrenean lineage of subterranean Trechini (Fig 4). The reduction to one ovariole has therefore occurred at least twice in Aphaenops. Other evidence that such a reduction occurred twice in the Pyrenean lineage is the presence of at least two ovarioles in the species of Geotrechus studied. Because the genus Geotrechus is not monophyletic [25], the regression in ovariole number in the two cave-adapted lineages of Trechini (eastern and western radiations) suggests an independent regressive evolution in the Pyrenean lineage. Unlike the Pyrenean species, the number of ovarioles is high in the Adriatic subterranean species Typhlotrechus bilimeki Sturm ([19]; Table 1). Typhlotrechus originated in the Oligocene and underwent little diversification (two species only) [21,24].

Many species of Duvalius are troglobitic and have undergone a reduction in the number of ovarioles, as in the subterranean species of the genus Trichaphaenops. This reduction is less drastic than in Aphaenops. Although troglobitic, they still possesses two ovarioles per ovary, as in non-troglomorphic representatives of Duvalius, but we do not know whether the species of Duvalius mentioned by Deleurance (Table 1) is a subterranean species or not, since it was not identified to species [19]. The Duvalius lineage diversified much more recently than the Pyrenean lineage, and the origin of the hypogean genus Trichaphaenops dates from the Plio- or Pleistocene, whereas the Pyrenean lineage originated in the Early Miocene [24]. This suggests a possible link between the age of the subterranean lineage and the degree of reduction in the number of ovarioles. The number of ovarioles within the genus Trechus varies from 3 to 7 according to the few species studied so far, but further investigations are required since this genus is very speciose, widespread and polyphyletic, with species colonizing various habitats, from high altitude mountains to subterranean environments [24, 68–69].