Interspecific Mating Behavior Between Introduced Zacco Platypus and Native Opsariichthys Evolans in Taiwan

Zoological Studies 59: 6 (2020) doi:10.6620/ZS.2020.59-06

Open Access

Interspecific Mating Behavior Between Introduced Zacco platypus and Native Opsariichthys evolans in Taiwan

Neng-Li Liao1,2, Shih-Pin Huang1, and Tzi-Yuan Wang1,*

1Biodiversity Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan. *Correspondence: E-mail: [email protected] (Wang). E-mail: [email protected] (Huang) 2Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan. E-mail: [email protected] (Liao)

Received 9 October 2018 / Accepted 22 January 2020 / Published 9 March 2020 Communicated by Hin-Kiu Mok

Introduced freshwater fishes considerably influence the ecology and populations of native species. Previous research has revealed that introduced Zacco platypus may hybridize with chubs that are sister but distinct genera. However, we have little knowledge of Z. platypus’ mate choice or its impact on Taiwanese chubs. Therefore, this study identified the interspecific mating behavior between introduced Z. platypus and native Opsariichthys evolans and evaluated the former’s invasive impact on cohabitants. Our observations showed that interspecific mating pairs do occur between Z. platypus male(s) and O. evolans female(s). Fifty-three percent of spawning events were interspecific mating and only 43% were between native O. evolans mating pairs. This study showed that Z. platypus male satellites might prefer to engage with Z. platypus, while O. evolans might engage by chance. However, introduced males of Z. platypus may be unable to recognize conspecific females. Meanwhile, introduced females of Z. platypus also have a mate choice preference for males of Z. platypus. Therefore, Z. platypus male hybridization might significantly reduce the successful mating ratio of O. evolans, leading to a dramatic reduction in native O. evolans offspring in the future. Key words: Spawning behavior, Chub, Mate choice, Opsariichthys, Zacco.

BACKGROUND among haplotypes ranged from 0.2% to 2.82%, and its high haplotype diversity implies multiple introduced The Opsariichthys group (Teleostei: Cypriniformes) populations from Japan. The origin of Z. platypus in contains common minnows found in freshwater Taiwan is questionable, although a previous study habitats, including the genera Parazacco, Candidia, suggests that it came from Lake Biwa, Japan (Ma et al. Nipponocypris, Zacco, and Opsariichthys (Huang et 2006). Nevertheless, the introduced Z. platypus may al. 2017). There are five species in the Opsariichthys greatly influence closely-related native Opsariichthys group in Taiwan—three endemic Opsariichthys species, species. Candidia barbata (Regan, 1908), and the introduced Introduced freshwater fishes have considerably Zacco platypus (Temminck & Schlegel, 1846) (Chen et influenced the ecology and populations of native species. al. 2008 2009; Huang et al. 2017; Kitanishi et al. 2016; However, we have little knowledge on Z. platypus’ Ma et al. 2006). Zacco platypus was identified in the mate choice or impact on Taiwanese chubs, although Tamsui River as an introduced species from Japan in the a similar mating behavior and reproductive process in 1980s (Ma et al. 2006). Its D-loop nucleotide divergence chubs was reported in Japan (Katano 1985 1990a b 1992

Citation: Liao NL, Huang SP, Wang TY. 2020. Interspecific mating behavior between introduced Zacco platypus and native Opsariichthys evolans in Taiwan. Zool Stud 59:6. doi:10.6620/ZS.2020.59-06.

1994 1998) and Taiwan (Chuang et al. 2006; Wang accident, mating, and/or stimulation. et al. 1995; Yan et al. 1995). For example, the mating This study reveals interspecific mating behavior behavior of Candidia barbata was reported as being between introduced Z. platypus and native O. evolans similar to that of the Japanese dark chub Nipponocypris and their mate choice; it shows how this interspecific temminckii (Yan et al. 1995). Their spawning mating may influence the population of native offspring behaviors are quite similar to those of N. temminckii: by reducing the rate of O. evolans intraspecific mating. a male presses a female laterally and stirs up the riverbed by vibrating its anal fin to bury released eggs (Katano 1983). Katano (1983) indicated two clearly- MATERIALS AND METHODS distinguished levels of social status in males: leading males that monopolize females and satellite males Study areas (sneakers) that dash toward spawning pairs. Leading males were the dominant individuals, but not always Two spawning areas with 7–19 redds in the the largest. In some cases, they established territories Keelung River were chosen as the study areas; both are around the spawning redd and, on one occasion, two located in Ruifang District, New Taipei City in northern leading males used the same spawning redd without Taiwan. Study site A (25°06'22.8"N, 121°48'33.0"E) is establishing territories. about 4 m long and 3 m wide, and has an average depth In addition, two sympatric Japanese chubs—Z. of 15 cm. Study site B (25°06'22.9"N, 121°48'32.2"E) is platypus and Nipponocypris temminckii—are known to about 8 m long and 5 m wide, and has an average depth have aggressive interactions during their reproductive of 13 cm. The water temperature is 26–36°C at both activities (Katano 1994). These two species commonly sites. occupy overlapping home ranges and occasionally defend a territory against other individuals. Furthermore, Ethogram the hybrids of Z. platypus and phylogenetically distinct Nipponocypris species—N. sieboldii (Temminck & A successful spawning event is defined as follows: Schlegel, 1846) and N. temminckii (Temminck & when spawning, both sexes remain close to each other Schlegel, 1846)—have been reported, which might be and continually shake their caudal fins, then lean to one due to artificial propagation, environmental temperature, side and the female lays its eggs (Wang et al. 1995; Yan and/or sympatric habitats increased by hydraulic et al. 1995). This study defined the starting point of a engineering (Arao and Shimoyama 2006; Katano et al. spawning event as when both sexes shake their caudal 2014; Kato 2004; Sakai et al. 1992). Thus, it is very fins (Table 1). After observing this shaking behavior, possible that the introduced Z. platypus hybridizes we continued to record the event until no other fishes with Taiwanese Opsariichthys species, especially the appeared in the redd. Such a successful spawning event cohabitated Opsariichthys evolans (Jordan & Evermann, was counted as an independent event. 1902). This study identified such interspecific mating behaviors and evaluated the former’s invasive impact Satellites on cohabitants. There are several distinguishing morphological Satellites are unpaired mature males (Table 1). features between Z. platypus and O. evolans—different These satellites always try to engage in cuckoldry to a length of pectoral fin ray, different patterns of pearl mating pair during spawning. The paired male always organs, and different body color patterns between followed with the paired female while the satellites the males; different stripe colors and snout tip colors swim around different mating pairs. between the females (details see Fig. 1; MATERIALS AND METHODS). A recent study further revealed that Species identification Z. platypus females choose males based on the degree of their red nuptial coloration, which suggests that There are several distinguishing morphological different body color patterns may influence mate choice features between Zacco platypus and Opsariichthys (Takahashi 2018). evolans (Jordan & Evermann, 1902): different length of Interspecific mating behavior has only been the pectoral fin ray, different patterns of pearl organs, systematically studied in several species of animals, and different body color patterns between the males (Fig. such as the leaf beetle, pipefish, and gecko (Peacock et 1a, c); and different stripe colors and snout tip colors al. 2004; Wilson 2006; Groning and Hochkirch 2008; between the females (Fig. 1b, d). Buden et al. 2014), and these cases were assigned as The body of O. evolans has a silver background parts of reproductive activities due to misidentification, with yellowish brown in the dorsal area, the body color

patterns have 11–13 vertical greenish-blue stripes in (2) a yellowish green caudal peduncle; mature females mature males, and shorter stripes in females (Chen do not have either character. In addition, parts of the et al. 2009). The pectoral fin is rather long, and its pectoral, dorsal, and anal fins turn red in mature males, maximum length reaches the pelvic fin base in mature and these fins are usually yellow in mature females. males, but the pectoral fin is clearly shorter in females. The body of Z. platypus also has a silver Sexual dimorphism can be clearly observed during the background and is yellowish-brown in the dorsal reproductive season (Fig. 1a, b). Mature males have (1) area. But the body color patterns have several vertical independent pearl organs on the cheek and snout and grayish-blue stripes, some of which stripes fuse to form

Fig. 1. Character identification of two species. a, O. evolans male; b, O. evolans female; c, Z. platypus male; and d, Z. platypus female. All morphological features for species identification were labeled using Arabic numerals and mentioned as follows. The mature male of O. evolans has: 1, 11–13 vertical greenish-blue stripes; 2, elongated pectoral fin; 3, independent pearl organs on the cheek and snout; 4, yellowish green caudal peduncle. The mature female of O. evolans has: 5, shorter vertical stripes; 6, usually grayish white snout tip. The mature male of Z. platypus has: 7, several vertical grayish-blue stripes and some of these stripes fuse into a wide bar; 8, medium length of pectoral fin; 9, aligned pearl organs on cheek and snout. The mature female of Z. platypus has: 10, a rather indistinct pale yellow or gray stripe; 11, usually orange red snout tip.

Table 1. Ethogram

Behavior Definition

Spawning event Both sexes remain close to each other and continually shake their caudal fins, then lean to one side and lay their eggs with disturbed sand. Cuckoldry Alternate male strategy; small males or satellite may engage in cuckoldry in species where spawning is dominated by large and aggressive males. Mating pair A large and/or aggressive male follows and cruises around a mature female during spawning. Territoriality During spawning, both sexes remain close to each other. The paired male circles around the redd and shows its fins to other approaching adults, and/or moves out to fight with the approaching individuals.

a wide bar in mature males. In addition, the stripes are disturbing the mating pairs (Fig. 2a, b, c). In addition, a rather indistinct pale yellow or gray in females. Its the digital handy cameras were also used to record pectoral fin is clearly shorter, and its maximum length chubs during the spawning event with a wider angle does not reach the pelvic fin base. Sexual dimorphism (Fig. 2d, e). We followed the paired male/female and can also be clearly observed during the reproductive recorded their spawning events via the digital handy season (Fig. 1c, d). Mature males have (1) aligned pearl cameras. All digital files were analyzed on a laboratory organs on their cheek and snout, and (2) the tip of their computer. snout is grayish black; mature females do not have To prevent the underwater housing cameras from pearl organs, and the tip of their snout is orange red. In interfering with the mating behavior, we first tested the addition, parts of the pectoral, ventral, dorsal, and anal working distance to the redd and determined whether fins turn red in mature males, and these fins are usually the small camera was too close to the redd. Although yellow in mature females. mature males that spotted the camera sometimes tried Females of the two species have fewer to attack it with their snout or looked for a different distinguishable characteristics than males (Fig. 1b, d). nearby redd, the cameras did not affect the paired chubs’ Nevertheless, two morphological features can still be spawning behavior in the redd (Fig. 2, video S1 and distinguished between the two female chubs. First, video S2). females of both species have several vertical stripes, but those of O. evolans are yellowish green and those Statistics of Z. platypus are a rather indistinct pale yellow or gray. Second, the snout tip is usually grayish white in O. The types of mating pairs and number of satellites evolans females, but orange red in Z. platypus females. were counted independently within a successful In addition, the pearl organs on the head and anal spawning event. In addition, if the species of an fin regions are also regarded as diagnostic features individual could not be recognized, then the event was for distinguishing a mature male from a female and discarded in our statistics. The mate choice preference subadult individual. Katano (1990b) showed that was determined by the Chi-square test of observed pearl organs on the head are a weapon in conspecific and expected spawning events. The Wilcoxon-Mann- aggressive encounters, and the pearl organ on the anal Whitney rank sum test was used to determine if fin is a tool for burying eggs during spawning seasons; satellites had a significant preference for any of the it is also highly associated with a large body size in three kinds of mating pairs. The Bonferroni multiple males. test was performed to evaluate if the false discovery rate (FDR) < 0.05. Types of mating pairs

Mature male and female chubs formed mating RESULTS pairs during the spawning season (Table 1). The paired chubs then searched for a redd to spawn in. There were Relative proportions of the two species four possible types of mating pairs: (a) O. evolans♂ × O. evolans♀; (b) Z. platypus♂ × Z. platypus♀; (c) To determine the putative proportions of offspring Z. platypus♂ × O. evolans♀; (d) O. evolans♂ × Z. from different types of mating pairs in the two species, platypus♀. The former two types were intraspecific we calculated the relative proportions of the two species and the latter two were interspecific. Furthermore, if from observed mating pairs. The two supplemental satellites from different species engaged in the former videos show intraspecific and interspecific mating two types, such a spawning event was also defined as events recorded by the handy and housing cameras. We interspecific mating. observed 78 spawning events over the spawning season, six of which were discarded because the species/ Video recording in habitats individuals could not be recognized. The 72 sampling events represented the types of mating pairs in our Digital handy cameras (Sony FDR-AXP55 and study. Cannon G16) and housing Polaroid CUBE+ cameras Of the individuals involved in the 72 recorded were used to record all mating behaviors. The spawning spawning events, 63 were male Z. platypus and 113 events were recorded in the studied habitat for five were male O. evolans (Table 2 and Table S1). On each independent days from 9:00 to 12:00 in July 2018 recording day, the paired male followed the paired (spawning season). The housing Polaroid CUBE+ female. Thus, the mating pair number was represented cameras were placed near the spawning redds without by the relative proportion of introduced Z. platypus

(a) (b)

(c)

(d) (e)

Fig. 2. The video clips from the housing Polaroid CUBE+ and handy cameras. (a) Intraspecific mating pair; (b) two paired chubs spawning nearby; (c) satellites trying to engage with paired chubs; (d) O. evolans mating; (e) Z. platypus mating. Clips (a–c) were captured on the housing camera. Clips (d– e) were captured on handy cameras.

Table 2. Total numbers of mating pair events and satellites

Satellite Spawning events Total number, involved events (%) Type of mating pair Observed Expected O. evolans♂ Z. platypus♂

O. evolans♂ × O. evolans♀ 39 (54%) 29.7 (41.2%) 37, 18 (45%) 9, 8 (38%) Z. platypus♂ × Z. platypus♀ 8 (11%) 9.2 (12.8%) 8, 5 (12%) 2,1 ( 5%) Z. platypus♂ × O. evolans♀ 24 (33%) 16.6 (23%) 28, 17 (43%) 20, 12 (57%) O. evolans♂ × Z. platypus♀ 1 (2%) 16.6 (23%) 0, 0 (0%) 0, 0 ( 0%)

Total number 72 (100%) 72.1 (100%) 73, 40(100%) 31, 21(100%)

(Zp) and native O. evolans (Oe) populations, which revealed that 35% of spawning events were from was around 35.8% and 64.2%, respectively. Based on interspecific mating pairs. Only 54% of spawning this ratio, the expected frequency of Oe × Oe pairing events were from native O. evolans mating pairs. In under the assumption of random mating should be addition, 8 of the 39 O. evolans spawning events were 0.642 × 0.642 = 41.2% (0.702 × 0.702 = 49.3%); Zp × engaged by Z. platypus satellites; 5 of the 8 Z. platypus Zp should be 0.358 × 0.358 = 12.8% (0.298 × 0.298 = spawning events were engaged by O. evolans satellites. 8.9%); and Oe × Zp and Zp × Oe should each be 23.0% Thus, 53% of the spawning events were interspecific (20.9%). Considering that the satellites still affect and only 43% were between native O. evolans mating offspring number, we also used the relative proportion pairs/satellites. of satellites in each species (29.8% of Z. platypus vs 70.2% of O. evolans), which showed no significant Mating preferences difference in subsequent analyses (data not shown). There might, however, be some bias around the relative Statistical analysis further revealed different proportion of the species, such as putative multiple mating preferences between males and females of counts, uncounted female numbers, and environmental both species, even when different relative proportions changes from human activities. were used (Table 3). Males of O. evolans significantly In addition, we also harvested some specimens for preferred mating with females of O. evolans, while future studies. The relative proportions of the species (Z. females only showed a slightly significant preference for platypus vs O. evolans) ranged from 50.0% vs 50.0% males. In contrast, females of Z. platypus significantly to 11.1% vs 88.9% (Table S2). Such variation might preferred males of Z. platypus, across different relative be due to insufficient sampling size and/or seasonality. proportions (Table 4). However, males of Z. platypus Nevertheless, the relative proportion of the two species showed no significant preference for females of one (11.1% of Z. platypus vs 88.9% of O. evolans) during species (Table 5), even though males of Z. platypus the spawning season was also used in subsequent were shown to slightly prefer females of O. evolans analyses. when we used the capture ratio (11.1% of Z. platypus vs 88.9% of O. evolans) as a relative proportion (p-value = Mating pairs 0.0123, data not shown). Such a slight difference could be due to the uneven population proportion between Four types of mating pairs were observed, but the females of both species. Thus, males of Z. platypus their ratios were not even (Table 2). Our observations might show no preference for females of either species.

Table 3. Mate choice preference via Chi-square test of observed and expected spawning events

O. evolans♂ Z. platypus♂ Z. platypus♂ O. evolans♂ × × × × O. evolans♀ Z. platypus♀ O. evolans♀ Z. platypus♀

O. evolans♂ × O. evolans♀ Z. platypus♂ × Z. platypus♀ 0.0784 Z. platypus♂ × O. evolans♀ 0.0122* 0.0613 O. evolans♂ × Z. platypus♀ 0.0000*** 0.0001*** 0.0000***

*p < 0.05, ***p < 0.005.

Table 4. Mate choice of the two female chubs

O. evolans ♀ Z. platypus ♀

Observed expected observed expected

O. evolans ♂ 39 40.45 1 5.78 Z. platypus ♂ 24 22.55 8 3.22

p-value 0.7039 8.93E-04****

****p < 0.001.

Satellite male engagement preference between in. Tables 8 and 9 further show that when the mating the species female is O. evolans, Z. platypus and O. evolans satellites prefer to engage in mating pairs with a male Table 6 indicates the number of satellite males of the same species, even using different relative that joined the mating process and table 7 presents the proportions. The satellites did not show any difference number of mating cases that satellite males engaged in preference in the other comparisons, although the

Table 5. Mate choice of the two male chubs

O. evolans ♂ Z. platypus ♂

Observed expected observed expected

O. evolans ♀ 39 25.68 24 20.54 Z. platypus ♀ 1 14.32 8 11.46 p-value 1.12E-05**** 0.2025

****p < 0.001.

Table 6. Average numbers of satellites within a spawning event

Satellite number mean ste (min, max) Type of mating pair ± O. evolans♂ Z. platypus♂

O. evolans♂ × O. evolans♀ 0.95 ± 0.21 (0, 5) 0.23 ± 0.08 (0, 2) Z. platypus♂ × Z. platypus♀ 1.00 ± 0.33 (0, 2) 0.25 ± 0.25 (0, 2) Z. platypus♂ × O. evolans♀ 1.12 ± 0.21 (0, 4) 0.80 ± 0.18 (0, 3)

Table 7. Satellite preference events

O. evolans satellite choice Z. platypus satellite choice Type of mating pair Observed event Expected event Observed event Expected event

O. evolans♂ × O. evolans♀ 18 (45%) 16.48 (41.2%) 8 (38%) 8.65 (41.2%) Z. platypus♂ × Z. platypus♀ 5 (12%) 5.12 (12.8%) 1 ( 5%) 2.69 (12.8%) Z. platypus♂ × O. evolans♀ 17 (43%) 9.20 (23%) 12 (57%) 4.83 (23%) O. evolans♂ × Z. platypus♀ 0 ( 0%) 9.20 (23%) 0 ( 0%) 4.83 (23%)

Total number 40(100%) 40 (100%) 21(100%) 21 (100%)

Table 8. Significance ofO. evolans satellite choice among four kinds of mating pairs via Chi-square test

O. evolans♂ Z. platypus♂ Z. platypus♂ O. evolans♂ p-value × × × × O. evolans♀ Z. platypus♀ O. evolans♀ Z. platypus♀

O. evolans♂ × O. evolans♀ Z. platypus♂× Z. platypus♀ 0.7053 Z. platypus♂ × O. evolans♀ 0.0094** 0.0101* O. evolans♂× Z. platypus♀ 0.0022*** 0.0024*** 0.0001***

*p < 0.05, **p < 0.01, ***p < 0.005.

results were statistically significant (Tables 8 and 9). the successful production rate of native O. evolans. Thus, satellite males of both species might engage with Meanwhile, introduced Z. platypus females also prefer their own species if they recognize mating males. to mate with males of Z. platypus. Thus, both situations may significantly decrease the populations of native O. evolans over time. DISCUSSION Furthermore, satellite preference between the two species revealed that the satellite males of both This study is the first to confirm that interspecific species might engage with their own species because mating behavior frequently happens between introduced they recognize conspecific mating males. As a result, Z. platypus and native O. evolans (35% in Table 2). Z. platypus satellites mainly engage with Z. platypus♂ Previous studies indicated mixed genetic diversity, × O. evolans♀ mating pairs (Tables 6 and 9). Such suggesting that introduced populations of Z. platypus satellite choice between the two species implies that Z. hybridize with native populations in Lake Biwa platypus males might be able to distinguish between (Takamura and Nakahara 2015) and very possibly with males of two species, while O. evolans female cannot. Taiwanese species in the Tamsui River (Ma et al. 2006). In other words, Z. platypus male satellites might prefer More and more hybrids of Z. platypus and distinct to engage with Z. platypus while O. evolans might Nipponocypris species have been reported in Japan over engage by chance. This bias may reduce the successful the years (Arao and Shimoyama 2006; Katano et al. rate of O. evolans intraspecific mating, and this is 2014; Kato 2004; Sakai et al. 1992). Thus, introduced supported by the observation that 35% of the spawning Z. platypus may also hybridize with closely-related O. events were interspecific. Thus, if reproductive isolation evolans and therefore decrease the populations of native remained in the two species and caused all hybrids chubs in Taiwan. to die, then the offspring number of O. evolans could A previous study indicated that Z. platypus females decrease by 35% every generation. On the other hand, choose males based on the degree to which they express offspring of Z. platypus could have a higher chance of red nuptial coloration, and do not look for traits related occupying O. evolans’ habitat niches. Therefore, one to the greenish-blue nuptial color of males. Such female can expect a sequential decrease and serial increase in O. preference may be useful for selecting high-quality evolans and Z. platypus, respectively. males with good foraging abilities or good immune functions related to carotenoids and sexual hormones (Takahashi 2018). Thus, closely-related O. evolans CONCLUSIONS might have a similar preference mating. Statistical analysis further revealed that native O. evolans prefer This study revealed that Z. platypus and putative native individuals (Table 3). In contrast, females of hybrids might have colonized the Tamsui River as Z. platypus significantly prefer males of Z. platypus, the result of previous fish fry releasing activities. across different relative proportions (Table 4). However, Fortunately, records show that introduced Z. platypus males of Z. platypus showed no significant preference only dispersed in the Tamsui River (Ma et al. 2006). To for females of either species. Therefore, this might lead avoid genetic pollution from reoccurring introgression to interspecific mating pairs, which in turn would lead events and to maintain the genetic diversity of native to hybrids (Table 5). This study implies that Z. platypus Opsariichthys fishes, it is of the utmost importance males may be unable to recognize conspecific females. that these Opsariichthys species are not artificially Thus, introduced Z. platypus males compete with released into river systems. Further studies should focus native O. evolans males for native females and reduce on their population distribution, effective population

Table 9. Significance ofZ. platypus satellite choice among four kinds of mating pairs via Chi-square test

O. evolans♂ Z. platypus♂ Z. platypus♂ O. evolans♂ p-value × × × × O. evolans♀ Z. platypus♀ O. evolans♀ Z. platypus♀

O. evolans♂ × O. evolans♀ Z. platypus♂ × Z. platypus♀ 0.2923 Z. platypus♂ × O. evolans♀ 0.0011*** 0.0006*** O. evolans♂ × Z. platypus♀ 0.0272* 0.0152* 0.0001***

*p < 0.05, ***p < 0.005.

size, and threat to native species to understand future Developments in environmental biology of fishes, pp. 165–183. conservation biology strategies around speciation, Springer, Dordrecht. doi:10.1007/s10641-009-9499-y. Chuang LC, Lin YS, Liang SH. 2006. Ecomorphological comparison hybridization, and genetic introgression between and habitat preference of 2 cyprinid fishes, Varicorhinus introduced Z. platypus and native Opsariichthys fishes. barbatulus and Candidia barbatus, in Hapen creek of northern Taiwan. Zool Stud 45:114–123. Acknowledgments: We thank Mr. Bo-Cyun Wang, Gröning J, Hochkirch A. 2008. Reproductive interference Miss Chia-Yu Lin, and Miss Geng-Xin Kong for their between animal species. Quart Rev Biol 83:257–281. doi:10.1086/590510. field assistance, and Mr. Noah Last of Third Draft Huang SP, Wang FY, Wang TY. 2017. Molecular phylogeny of Editing for his English language editing. This study the Opsariichthys group (Teleostei: Cypriniformes) based on was funded by the Ministry of Science and Technology, complete mitochondrial genomes. Zool Stud 56:40. doi:10.6620/ Taiwan (MOST 105-2311-B-001-064, MOST 106-2311- ZS.2017.56-40. B-001-022, MOST 107-2311-B-001-007). Katano O. 1983. Spawning of the dark chub, Zacco temmincki (Temminck et Schlegel) (Pisces: Cyprinidae), with special reference to male social status. Physiol Ecol Japan 20:33–52. Authors’ contributions: SPH and TYW designed Katano O. 1985. Aggressive-behavior and dominance relationships of the study and prepared the manuscript. NLL, SPH, and the dark chub, Zacco temmincki with special reference to their TYW performed the field work and video analysis. individual recognition. Japanese J Ichthyol 32:225–238. All authors participated in revising the manuscript and Katano O. 1990a. Dynamic relationships between the dominance of male dark chub, Zacco temmincki, and their acquisition of approved the final manuscript. females. Anim Behav 40:1018–1034. Katano O. 1990b. Seasonal, sexual and individual variations in gonad Competing interests: NLL, SPH, and TYW declare weight and secondary sexual characters of the dark chub, Zacco that they have no conflict of interest. TYW has received temmincki. Japanese J Ichthyol 37:246–255. research grants from the MOST, Taiwan. Katano O. 1992. Spawning tactics of paired males of the dark chub, Zacco temmincki, reflect potential fitness costs of satellites. Environ Biol Fish 35:343–350. Availability of data and materials: There are Katano O. 1994. Aggressive interactions between the dark chub, two supplementary videos showing intraspecific and Zacco temmincki, and the pale chub, Zacco platypus, in relation interspecific mating behaviors. supplement-vedio_cube. to their feeding behavior. Japanese J Ichthyol 40:441–449. avi (video S1) was recorded on the underwater housing Katano O. 1998. Growth of dark chub, Zacco temmincki (Cyprinidae), with a discussion of sexual size differences. Environ Biol Fish camera; supplement-vedio_handy-cam.avi (video S2) 52:305–312. was recorded on the handy camera. Katano O, Baba Y, Ohara H, Kawamura K, Sato M, Kumagaya M, Takeuchi M, Ito S, Togashi S, Inoue N. 2014. Enlarged Consent for publication: All authors have approved distribution of Nipponocypris temminckii as a domestic alien the manuscript and agree with its submission to fish. Japanese J Ichthyol61: 97–103. Kato F. 2004. Geographic distribution of Zacco (Cyprinidae) and a Zoological Studies. record of hybrid in Fukui prefecture, Japan. Bull Fukui City Mus Nat Hist 51:15–24. Ethics approval consent to participate: Not Kitanishi S, Hayakawa A, Takamura K, Nakajima J, Kawaguchi Y, applicable. Onikura N, Mukai T. 2016. Phylogeography of Opsariichthys platypus in Japan based on mitochondrial DNA sequences. Ichthyol Res 63:506–518. doi:10.1007/s10228-016-0522-y. Ma GC, Watanabe K, Tsao HS, Yu HT. 2006. Mitochondrial REFERENCES phylogeny reveals the artificial introduction of the pale chub Zacco platypus (Cyprinidae) in Taiwan. Ichthyol Res 53:323– Arao K, Shimoyama J. 2006. Hybrids between Zacco platypus and Z. 329. doi:10.1007/s10228-006-0353-3. temminckii from Aichi prefecture. Japan Sci Rep Toyohashi Mus Peacock L, Batley J, Dungait J, Barker JHA, Powers S, Karp Nat Hist 16:53–54. A. 2004. A comparative study of interspecies mating of Buden DW, Cianchini C, Taborosi D, Fisher RN, Bauers AM, Phratora vulgatissima and P. vitellinae using behavioural Ineich I. 2014. Photographic evidence of interspecies mating tests and molecular markers. Entomol Exp Appl 110:231–241. in geckos of the lepidodactylus lugubris unisexual-bisexual doi:10.1111/j.0013-8703.2004.00143.x. complex (Squamata: Gekkonidae). Phyllomedusa 13:133–136. Sakai H, Nagata A, Fujioka Y. 1992. Geographic distribution of Zacco doi:10.11606/issn.2316-9079.v13i2p133-136. (Cyprinidae) and occurrence of hybrids in Yamaguchi prefecture. Chen IS, Wu JH, Hsu CH. 2008. The taxonomy and phylogeny of the J Shimonoseki Univ Fish 40:75–82. cyprinid genus, Candidia (Teleostei: Cyprinidae) from Taiwan, Takahashi D. 2018. Female mate choice based on male nuptial with description of a new species and comments on a new genus. coloration in pale chub, Zacco platypus. Zoolog Sci 35:23–27. Raffles Bull Zool Suppl19: 203–214. doi:10.2108/zs170102. Chen IS, Wu JH, Huang SP. 2009. The taxonomy and phylogeny of Takamura K, Nakahara M. 2015. Intraspecific invasion occurring in the cyprinid genus Opsariichthys bleeker (Teleostei: Cyprinidae) geographically isolated populations of the japanese cyprinid fish from Taiwan, with description of a new species. In: D. L. G. Zacco platypus. Limnol 16:161–170. doi:10.1007/s10201-015- Noakes, A. Romero, Y. Zhao and Y. Zhou (eds.) Chinese fishes. 0450-y.

Wang JT, Liu MC, Fang LS. 1995. The reproductive biology of an Supplementary materials endemic cyprinid, Zacco pachycephalus, in Taiwan. Environ Biol Fish 43:135–143. Wilson AB. 2006. Interspecies mating in sympatric species of Table S1. Spawning events observed on each sampling Syngnathus pipefish. Mol Ecol 15:809–824. doi:10.1111/j.1365- day. (download) 294X.2006.02831.x. Yan JH, Lue KY, Chen YS, Jeng JK. 1995. The study on the Table S2. The captured individuals of Z. platypus and reproductive ecology of Zacco barbata in Ha-Pen Creek. Biol O. evolans. (download) Bull Nat Taiwan Norm Univ 30:69–81. Video S1. Cube.avi. (download)

Video S2. Handy-cam.avi. (download)