Co-occurrence of ecologically equivalent cryptic species of spider wasps

Many cryptic species have been discovered in various taxonomic groups based on molecular phylogenetic analyses and mating experiments. Some sympatric cryptic species share equivalent resources, which contradicts the competitive exclusion principle. Two major theories have been proposed to explain the apparent lack of competitive exclusion, i.e. niche-based coexistence and neutral model, but a conclusive explanation is lacking. Here, we report the co-occurrence of cryptic spider wasp species appearing to be ecologically equivalent. Molecular phylogenetic analyses and mating experiments revealed that three phylogenetically closely related species are found sympatrically in Japan. These species share the same resources for larval food, and two of the species have the same niche for nesting sites, indicating a lack of competitive exclusion. This evidence may suggest that ecologically equivalent species can co-occur stably if their shared resources are sufficiently abundant that they cannot be over-exploited.


Molecular phylogenetic analyses
Haplotypes of each dataset and pairwise-distances among them are shown in table S7 and S8. In the BI and ML trees of the COI dataset (figure S2), the EN and WM types are not separated into monophyletic groups. In the COI dataset, maximum intratypic pairwise distance of the EN type was greater than the minimum pairwise distance between the WM and EN types (table S6). In contrast, the results of phylogenetic analyses based on the 28S dataset and mating experiments showed that they have been segregated at the species level. This contradiction suggests the possibility of mitochondrial gene introgression or incorrect phylogenetic relationships. Another study of Pompilidae also shows that the COI barcording region may produce misleading phylogenetic relationships [42].
The topology of the BI and ML trees of the 28S dataset is similar to that of the BI and ML trees of the combined dataset and suggests that the WM, BM and EN types (and also the OI, K1, K2, E1 and E2 types) are separated into each monophyletic group (figure S3).
We should also note that there are few haplotypes in all three species (Table S5), even though a fast region in COI is included in the current sequencing. Among them, the BM type is almost uniform: one sample (one haplotype) is different at one base pair from the other common haplotype, even though samples are collected widely from Hokkaido to Kyushu in Japan. Because samples are limited in corrected localities, this result may be due to the deviation of samples. We need further studies to find the reason for the scarcity of haplotypes in this species complex.

Mating experiments
(1) Normal mating behavior of each type Between a female and a male of the same type, the following behavioral sequence was observed (figure S4 and Supplementary Movie S1): the male moved toward the female, raising the metasoma and vibrating the wings, and then mounted on her dorsum ("courtship behavior"). In almost all courtships of the WM type wasps (44/50), the male rushed to the female without wing vibration. In many courtships of the BM type wasps (93/127), however, the male directly leaped on the female, vibrating the wings. In all EN type courtships, the male rushed toward the female, and many of them vibrated the wings (13/19). Next, the male lowered its metasoma on the female, vibrating both wings and antennae, and attempted to insert the genitalia into the female vagina. The male repeated this action many times until successful copulation. When the male genitalia were inserted, the female stopped moving for a while ("freezing"). Most males, after copulation, exhibited further courtship behavior toward the females. The latter, however, drove off the former ("mate rejection") and a second copulation never occurred.
(2) Examination of sperms In the females that had exhibited freezing (WM type: n = 24; BM type: n = 64; EN type: n = 1), sperm was found within the spermathecae. However, females not exhibiting freezing never had sperm (WM type: n = 3; BM type: n = 7). Based on this, we regarded other females exhibiting freezing as successful in mating.
(3) The frequency of male and female mating Females of this species complex exhibit single mating in their lifetime as many other solitary wasps and bees [43]. All females having copulated exhibited mate rejection and never copulated again even though the same type of males courted (WM type: n = 4; BM type: n = 20). In contrast, males often copulated multiple times (up to three times; WM type: n = 8; BM type: n = 24; EN type: n = 4).
(4) Characteristic mating behavior of each type Some differences in mating behavior were found among the types.
(4-1) Mounting time The WM and EN type males mounted on the same type females and kept the position more than one minute until they were driven off (mounting time: WM type, 141.7 sec., 62-238, SD = 46.3, n = 12; EN type, 131.9 sec., 72-258, SD = 64.1, n = 8). In contrast, the BM type males continued mounting for only several seconds (4.5 sec., 3-7, SD = 1.1, n = 17). The mounting time of the BM type was much shorter than that of the WM and EN types.
(4-2) Female's mate rejection behavior The female's mate rejection behavior also varied among the types. Both the WM and EN type females rapidly turned around several times in situ when the same type males exhibited courtship behavior (Supplementary Movie S2). In contrast, the BM type females never turned around but moved away from the same type males or drove them off with the legs.
The "turning-around" behavior of virgin females was also found in the following heterotypic pairs: the WM type female and EN type male; and the EN type female and WM type male. This behavior was, however, never found when the BM type males courted the WM or EN type females.
(5) Female's mate rejection between different types Males occasionally attempted to copulate with other type females and all of them failed (courtship: WM type, n = 9; BM type, n = 23; EN type, n = 9; mounting: WM type, n = 5; BM type, n = 10; EN type, n = 2). When the males mounted the females and attempted to insert their genitalia, the females appeared to close their metasomal apices (figure S7). In contrast, all type females opened it to copulate with the same type males.

Proximate cause of reproductive isolation
Because the rate of the male courtship between the same type wasps was significantly higher than that between the different type wasps, the cause of the reproductive isolation of the WM, BM and EN types is interpreted proximately as the male's selection of the same type female. It is unclear what factors caused the male's selection. It is likely the male was not able to distinguish female types by morphological or behavioral cues. Because all type females have almost the same morphology, including color, and females didn't appear to exhibit any special behavior before male's courtship. Males are likely to use invisible cues for the perception of the same type females. It is known that many cryptic animal species use nonvisual mating signals [1] and that many hymenopterous species use sex pheromones [43].
It is also probable that the female discriminates the same type male from different type males. Although some males courted other type females, they never copulated. Even when males mounted on the other type females and attempted to insert their genitalia, they never succeeded. When the female perceives the same type male, it may also be that the male sends out some signals to the female. Probably the signals are nonvisual because the WM and EN type males are very similar morphologically. There is a possibility that the male uses chemical signals. Male pompilids have the "Day's organ", which is situated on both the intersegmental membranes and the metasomal terga and is suspected to be secretory organs of some sex pheromones [18,44]. Some males were observed to raise the metasoma (figure S4A), extend its segments and expose the intersegmental membranes when they courted. They may emit some pheromones from the organs and send them out by wing vibration. Alternatively, they might make courtship sounds with the wings as certain pteromalid and braconid males [45,46]. It is, however, noteworthy that most males of the WM type courted without wing vibration. Because copulation never occurred when females were courted by heterotypic males, a female's mate selection is assumed to be more accurate than that of the male. Fig. S1: Head of Auplopus carbonarius species complex, anterior view (A-K) and whole body of its female (L). (A) White mandibular type male; (B) black mandibular type male; (C) exposed nesting type male; (D) White mandibular type female; (E) black mandibular type female; (F) exposed nesting type female; (G) Okinawa Island type male; (H) Korean type 1 male; (I) Korean type 2 male; (J) European type 1 male; (K) European type 2 male. Scales: 0.5 mm for (A-K); 5 mm for (L).

Supplementary Fig. S4: Mating behavior of Auplopus carbonarius species complex (black mandibular type). (A)
Courtship behavior-1, male moving toward female, raising metasoma and vibrating wings; (B) courtship behavior-2, male mounting on female dorsum and attempting to insert genitalia into female vagina; (C) male inserting genitalia.; (D) mate rejection, female driving off male exhibiting further courtship behavior after copulation.

Supplementary Fig. S5: Statistical tests for mating experiments. (A)
The copulation success of males with conspecific females is significantly more frequent than that with non-conspecific females (*, P < 0.0000001; **, P < 0.000000000001 in Fisher's exact test with Bonferroni correction). (B) The courtship of males toward conspecific females is significantly more frequent than that toward non-conspecific females (*, P < 0.00000001; **, P < 0.0000000000001 in Fisher's exact test with Bonferroni correction).

Supplementary Fig. S6: Per-month frequency distributions of wasps of Auplopus carbonarius species complex emerging from bamboo tube traps collected in winter.
All wasps were considered the first generation. WM: White mandibular type male; BM: black mandibular type male; EN: exposed nesting type male. The main emergence period of all three type wasps was April-May. Supplementary Fig. S7: Female's mate rejection to different type males. (A) A black mandibular type female mounted by a white mandibular type male, the latter attempting to insert his genitalia and the former appearing to close her metasomal apex; (B) white mandibular type female opening their metasomal apex to copulate with the same type male.