(a) Hybrid alate morphology from the alcohol-preserved samples
Three morphotypes were recognized among Taiwanese Coptotermes, from the 2019 survey and NCHU collection: C. formosanus (Fig. 2A), C. gestroi (Fig. 2B), which both conformed with their respective species description (Scheffrahn and Su 2000, Li 2010), and atypical individuals (Fig. 2C). The individual morphological measurements were plotted in Fig. 2D. Alates of C. formosanus are light brown in head color (sum of the RGB values, 197.53±32.79), and their antennal spots, if visible, are oblong and dim (Euclidean distance between head and spots, 45.78±13.12), with blurred borders (Fig. 2E). In comparison, alates of C. gestroi are dark brown in head color (RGB sum, 116.75±28.33), which contrast drastically with light colored and crescent-shaped antennal spots (Euclidean distance, 76.19±11.78), forming a defined border (Fig. 2F). Atypical alates did not fit either of the above descriptions. They displayed a relatively light head color like C. formosanus (RGB sum, 184.29±14.96, p=0.08) but antennal spots were apparent like C. gestroi (Euclidean distance, 81.45±8.59), which were numerically even more apparent than both C. gestroi (p=0.03) and C. formosanus (p<0.001). In addition, the antennal spots of atypical alates (0.102±0.005 mm) were thicker than those of C. gestroi (0.072±0.005 mm, Fig. 2G). Because of the differences of these atypical alates compared to C. formosanus and C. gestroi, we labeled them as “putative hybrid”.
In 2019, 2020, and 2021, Coptotermes alates were observed for 24, 19, and 13 nights, respectively. Additionally, dispersal flight events for C. formosanus, C. gestroi, and putative hybrids were observed for 39, 30, and 15 nights, respectively, resulting in the collection of 9,829 individuals of C. formosanus, 16,556 individuals of C. gestroi, and 1,218 individuals of putative hybrids. Most of the hybrid alates collected in this study were females, 1:9.9 (male: female). Of the 682 sampling events preserved in the NCHU Termite Collection, 553 were identified as C. formosanus, 128 were identified as C. gestroi, and only one was identified as a putative hybrid, with the exact number of individuals not recorded.
(b) Genetic analysis
Five loci, CopF6F, CopF10F, Cg33, CopF10-4, and Clac1 were amplified in C. formosanus alates (115/115, 100%), in C. gestroi (103/108, 95.3%, and in the putative hybrids were 31/32 (96.8%). Only the individuals which had all loci successfully amplified were used to perform STRUCTURE analysis. In the STRUCTURE analysis the best number of population clusters K=2 (Fig. S5). The clustering result indicated an obvious association between morphotype and genotype (Fig. 3). All individuals with C. formosanus morphology carried C. formosanus specific mitochondria were assigned to the same cluster (Fig. 3, the proportion of color gray >0.9); and all individuals in the C. gestroi morpho-type carrying C. gestroi specific mitochondria were assigned to the second cluster (Fig. 3, the proportion of color black >0.9). The individuals bearing atypical morphological appearance (the putative hybrids) were assigned as hybrid which displayed admixed composition with qi value >0.3 on both clusters. The evidence that their mitochondrial genotype were from either C. formosanus or C. gestroi further support their hybrid status from either heterospecific mating combination.
Private allele, alleles that are found only in a single population among a broader collection of populations, were sorted out (Table S2). The hybrid generation was inferred by identifying alleles inherited from C. formosanus and C. gestroi (Fig. S6). For a given locus, the first generation (F1) hybrids are obligated heterozygous for species-specific alleles. If two alleles were specific to the same species, one can rule out the possibility of such an individual as an F1 hybrid. By inspecting all the allele combinations of the 31 hybrids, four individuals were classified as “hybrids of more than the first generation (>F1)” (Fig. 3, Fig. S6).
(c) Dispersal flight events and time series curves
The dispersal flight events in the three consecutive monitored years (2019-2021) were recorded, and the average number of dispersal flight events at six defined time periods (early April, mid April, late April, early May, mid May, and late May) were counted (Fig. 4A). Dispersal flight models were obtained by fitting the Gaussian function curves (Fig. 4B). For the hybrids and the two parental species, each curve exhibited a single peak and had a shape that suggested a normal distribution (for all parameters, p-values < 0.05). The occurrence of C. gestroi reach the peak around late April; followed by the hybrids reaching the peak around early May, and C. formosanus reach the peak around mid May (Fig. 4B). Hybrid alates were frequently observed during simultaneous dispersal flights of either parental species: among the 15 times of hybrid dispersal flight events, 13 times (87%) were concurrent with either or both parental species.
(d) Reproductive performance examination
In the years 2021 and 2022, all possible mating combinations were established as incipient colonies in individual rearing units from live alates collected from field dispersal flight events (details for incipient colony replication for each mating combination were provided in Table S3). Before pairing (Fig. 5C), C. formosanus females harbored the most matured ovarioles (13.7±1.4), followed by C. gestroi females (11.6±1.2, p=0.003), while hybrid females harbored only 1 or no matured ovarioles (p<0.001, comparing with both species). After 8 days of rearing (Fig. 5A), colonies produced eggs but none had yet hatched. CF×CF colonies (13.9±1.7) produced significantly more eggs than CG×CG colonies (8.3±2.1). The F1 colonies developed as well as their maternal species; CF×CF and CF×CG had a similar number of eggs (p=1), and so did CG×CG and CG×CF (p=1). Backcross between hybrid males and parental species produced a less or equal number of eggs than the conspecific colonies (between CF×HY and CF×CF, p<0.001; between CG×HY and CG×CG, p=1). Colonies led by hybrid females (HY×HY, HY×CF, HY×CG) developed the worst, they produced less than 1 egg on average (ranging from 0 to 3).
After 54 days of rearing (Fig. 5D), the number of matured ovarioles of hybrid females was still less than 1 on average (ranging from 0 to 4), and was significantly lower (p<0.001) than that of C. formosanus females (16.3±1.9) and C. gestroi females (14.4±2.5). Eggs hatched and grew into larvae, workers, or soldiers after 54-day rearing. CF×CF colonies (20.5±8.2) produced significantly more eggs than CG×CG colonies (13.7±7.0, p<0.001). The F1 colonies developed as well as their maternal species; CF×CF and CF×CG had a similar number of offspring (p=1), and so did CG×CG and CG×CF (p=1). Although developing embryos were observed in the colonies of hybrid male backcrosses (CF×HY and CG×HY, Fig. S8, E, and F), only a few CF×HY colonies (7/22) had their eggs hatched (offspring number ranged from 1 to 22). Finally, none of the eggs produced by hybrid females hatched and no developing embryos were observed after 54 days of rearing (Fig. 5A).
The few eggs laid by hybrid females were malformed and unviable. Compared to the bean-like eggs found in the colonies led by C. formosanus and C. gestroi females (Fig. S7, A, and B; Fig. S8, A, D, E, F), the shape of eggs produced by hybrid females were highly variable. They varied from elongated-bean shape, spherical to short oval, and these eggs were usually smaller in volume than those laid by C. formosanus and C. gestroi (Fig. S7, C-U). Eggs laid by C. formosanus and C. gestroi females contained numerous evenly distributed small yolk granules and lipid droplets. After one week, the developing embryo could directly be observed when laid by a parental species female (Fig. S8, A, D, E, F). However, hybrid-female-produced eggs were irregular in internal structure, the yolk or lipid spheres were unevenly distributed and obviously larger than those of parental female species-produced ones (Fig. S8, B, and C).
Summing up the above findings, the colonies led by the females of either parental species grew rapidly with the emergence of workers and soldiers, both in conspecific (CF×CF, CG×CG) and heterospecific (CF×CG, CG×CF) mating combinations. In comparison, only 7 in a total of 22 colonies of hybrid males backcross C. formosanus females could develop and produce offspring, and all hybrid males backcross C. gestroi females failed. Hybrid females displayed abnormal ovariole morphology and produced oocytes that seldom developed further than stage II. Even if the eggs were laid, their internal structure was irregular, leading to embryos' nonviability.