Jpn. J. Environ. Entomol. Zool. 29（4）： 135 － 141 （2018） 環動昆 第 29 巻 第 4 号：135 － 141 （2018）

Original Article

Age class structure of spawning populations of two endangered endemic striated spined loaches in Lake Biwa river system

Kiyohito Morii1)* and Koh-Ichi Takakura2)

1) Graduate School of Environmental Science, The University of Shiga Prefecture, 2500, Hassaka-cho, Hikone-City, Shiga 522-8533, Japan 2) Environmental Science, The University of Shiga Prefecture, 2500, Hassaka-cho, Hikone-City, Shiga 522-8533, Japan

（Received: March 28 , 2018；Accepted: October 20, 2018）

Abstract Population dynamics models with age structure have often been applied to many organism species. Knowledge related to the age class structure, which is fundamentally important for such models, is expected to contribute to the effective conservation of endangered species. Two endemic spined loaches in the Lake Biwa river system, Cobitis magnostriata and C. minamorii oumiensis, have narrowed their distributions. Today, they are regarded as endangered species. For this study, to estimate their age structures, we surveyed body lengths of fish of the two species at a spawning site in Takashima, Shiga Prefecture for three years (2015–2017). A series of surveys captured 601 C. magnostriata individuals (488 male, 113 female) and 148 C. minamorii oumiensis individuals (60 male, 88 female). We applied models with different numbers of cohorts, k (1–7) for each sex and species and selected the best-fitted model among them. Models with k = 4 and k = 2 were selected respectively for C. magnostriata males and females. Models with k = 3 and k = 6 were selected respectively for C. minamorii oumiensis males and females. Considering the largest number of age classes in females of C. minamorii oumiensis and some studies related to this and related species, it is probable that females of this species have often failed to spawn and have repeatedly re-run to the spawning site, at least during these several years.

Keywords: age class structure, Cobitis, conservation, population ecology, reproduction, spawning

Introduction an age class structure is expected to contribute greatly to the efficient conservation of endangered species, even if it is rather Age class structure is an important determinant of incomplete. population dynamics, as emphasized in some prominent The Cobitis striata species complex (Cypriniformes, mathematical models such as the Leslie model (Leslie, 1945). Cobitidae) is a group of freshwater fishes in Japan with five Population dynamics models considering the age class species and 11 subspecies (Nakajima, 2017). The taxonomy in structure, such as the Leslie matrix model and Lefkovitch this species complex has been confused, although some matrix model, have often been used for the resource estimation regional races were reported (Minamori, 1955; Saitoh and of commercially important species in fisheries and for Aizawa, 1987) before the current taxonomy (Nakajima, 2012). sustainable analysis of endangered species (Ariga et al., 2014; All of them are listed as Critically Endangered, Endangered, or Watanabe et al., 2014). Determination of the age class structure Vulnerable (CR, EN or VU) on Japan’s red list (Ministry of the requires great effort. Therefore, it should generally be difficult Environment, 2015). Therefore, conservation of these for many species, especially for endangered species of which species/subspecies is a necessity. Nevertheless, the life history the population size has already become smaller. However, such of Cobitis striata species complex remains unclear. Surveys of

*Corresponding author: [email protected]

- 135 - Morii and Takakura spawning populations have not been conducted even the numbers of age classes. Based on those results, we qualitatively, but some anecdotal reports have been submitted demonstrated that C. minamorii oumiensis deserve deep for C. striata striata Nakajima, 2012 and C. minamorii concern for the future persistence of the population. minamorii Nakajima, 2012 (Saitoh, 1990). Both C. magnostriata Nakajima, 2012 and C. minamorii Materials and Methods oumiensis Nakajima, 2012 are endemic species distributed throughout the Lake Biwa river system of Japan. Before Study site Nakajima (2012) described C. magnostriata and C. minamorii We conducted a series of surveys at a fallow paddy field in oumiensis as new species, the former and latter species were Shin-Asahi, Takashima, Shiga Prefecture, Japan. Adults of both designated respectively as large race and Biwa-small race in C. magnostriata and C. minamorii oumiensis were found at the reports. They have mutually similar morphology (Saitoh, this site (Nakano et al., 2015). At the site was a ditch of ca. 1984), but C. magnostriata is larger than C. minamorii 230 m total length and 30–200 cm width. The depth was ca. 15 oumiensis in body length and egg size (Minamori, 1956). cm at most parts of the ditch and ca. 30 cm deep at the deepest Saitoh and Matsuda (1990) reported that adults of C. part. Details of the study site were presented by Morii et al. magnostriata inhabit Lake Biwa and spend most of their life (2018b). there, except for the spawning season. During May–June, they migrate to and spawn at small and permanent water areas Collection of adult fish and body length measurement connected to the lake, such as ditches and side streams. Saitoh Adult fish were collected at the study site using three and Matsuda (1990) also reported that adults of C. minamorii collection methods: hand-netting, traps targeting immigrating oumiensis inhabit ditches connecting Lake Biwa and the lake, individuals into the study site, and traps targeting emigrating and that they run to and spawn at temporal water areas such as individuals from the site. Both C. magnostriata and C. paddy fields during June–July: the two species spawn at water minamorii oumiensis live most of the year in Lake Biwa. The areas of different types. A recent report (Nakano et al., 2015), exception is the spawning season, in which sexually mature however, has described that both species spawned individuals migrate to ditches or paddy fields. Therefore, sympatrically at a site. individuals captured in this study are either sexually mature Earlier reports of the literature (Saitoh, 1993; Saitoh and individuals or newborn juveniles that were produced by them. Matsuda, 1990) suggested that C. magnostriata and C. Surveys were conducted while water flowed in the ditch: minamorii oumiensis were distributed throughout the coast of during May 9 – July 4 in 2015, May 10 – July 29 in 2016, and Lake Biwa. The distribution ranges of both species have April 22 – July 23 in 2017. All three collection methods were declined to the present day (Saitoh, 1993). Particularly, the used once a week in 2015. In 2016 and 2017, however, they distribution range of C. minamorii oumiensis appears to be were done once every two weeks in May–June and once a extremely smaller because all surveys of this species published week in the other duration. We conducted a total of 55 during this decade (Nakano et al., 2015; Morii et al., 2018a, hand-nettings on each survey day. The locations for 2018b) were conducted at only one site. No other spawning hand-netting were scattered. They covered the entire ditch site is known. Some conservation activities have been homogeneously. Hand-nettings were conducted by one person conducted at the site (Nakajima, 2017), but our field survey using a hand-net (38 × 38 cm, 1 mm mesh size). The body strongly suggests that C. minamorii oumiensis is still length was measured by placing the fish in an acrylic narrow endangered (Morii et al., 2018a). Therefore, clarifying the tank with a scale (AK-1, Mitani Tsurigyogu, Kohnosu). actual situation of the reproduction of both species is an urgent In addition to hand-netting, a small trap net (mesh size 4 task. The age structure is expected to contribute to our mm) was placed, respectively, at the inlet and outlet of the understanding of the current reproductive situations in the two ditch. At 18:00–19:00 of the day before the hand-netting species. survey, these traps were placed to capture adult fish migrating This study was conducted to clarify the age structure of from the canal into the ditch. Adults were collected in the traps spawning populations of both species in the field. We used at 8:00–9:00 of the next day. We covered the trap entrances hand-netting and trapping to collect adult fish to investigate the with a coarse net (mesh size 40 mm) to prevent Japanese size distributions for the respective species. Then we estimated common catfish, Silurus asotus Linnaeus, 1758, from entering

- 136 - Age class structure of spawning populations of two endangered endemic striated spined loaches in Lake Biwa river system

morphological characteristics. Multiplex polymerase chain reaction (PCR) using species-specific primers was used, targeting the mitochondrial DNA sequence. Details of molecular identification were explained in our earlier work (Morii et al., 2018b).

Statistical analysis We estimated the number of cohorts in the spawning populations of both species based on the size–frequency distributions of captured individuals. To do so, we estimated the number of components (single normal distributions) of the observed size frequency distribution. We regarded it as the Fig. 1 Small trap net used to collect fish that emigrated from number of age classes. Such estimation has often been done in the study site with drainage water. some fishes (Carlson et al., 2011; Frost, 1945).

The component number was estimated from the model the traps and preying on the fish. selection. Models with k (= 1–7) components were fitted to Using traps, we collected individuals of the two species that each observed size frequency distribution. A model with the fell from the study site when the water of the ditch at the site least Akaike’s information criteria (AIC) was selected as the was drained on July 4, 2015, July 2, 2016, July 29, 2016, June best-fitted model. For this estimation, all data obtained during 30, 2017, and July 23, 2017. We set small trap nets (1 mm three years were pooled, but the data were analyzed separately mesh size) at the ditch inlet and outlet, respectively (Fig. 1). by sex and species. For these analyses, the mixtools package of The survey was administered as the drainage water flowed R (ver. 3.3.1) was used. down from the ditch: it started at 9:00 and ended by 19:00. We recorded species and sex of captured individuals. The sex of Results each parent fish was distinguished based on the presence or absence of the lamina circularis; the lamina circularis is a In a series of surveys, 601 C. magnostriata individuals (488 plate-like structure on the breast fin. It is a trait specific to male, 113 female) and 148 C. minamorii oumiensis individuals males. For some individuals that could not be identified (60 male, 88 female) were captured. The respective body definitively, a part of the anal fin was cut off and preserved in lengths of C. magnostriata males and females were 54–94 and 99% ethanol. The 99% ethanol specimens were brought to the 65–109 mm (Fig. 2). The respective body lengths of C. laboratory at The University of Shiga Prefecture and were minamorii oumiensis males and females were 38–78 mm and stored at about 4°C for later analysis. Some individuals were 55–98 mm (Fig. 3). The size frequency distributions of species identified using molecular biological techniques, although and sexes were not monomodal. most captured individuals were identified based on

50 12 M ale Female 10 40 8

30 6 20 4

10 2 Number individuals of

0 0

65 68 71 74 77 80 83 86 89 92 95 98 54 57 60 63 66 69 72 75 78 81 84 87 90 93

101 104 107

Body length (mm)

Fig. 2 Frequency distributions of body length in C. magnostriata males (left) and females (right). Vertical lines in panels represent the positions of peaks of component normal distributions.

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8 8 M ale Female

6 6

4 4

2 2

Number individuals of

0 0

38 41 44 47 50 53 56 59 62 65 68 71 74 77 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 Body length (mm)

Fig. 3 Frequency distributions of body length in C. minamorii oumiensis males (left) and females (right). Vertical lines in panels represent the positions of peaks of component normal distributions.

Table 1 Results of model selections among a series of ks, the numbers of cohorts (component normal distributions) in the frequency distributions of body length, in both sexes of C. magnostriata and C. minamorii oumiensis. For the top four

models, parameter k (the number of components constructing the distributions), Akaike’s information criteria (AIC),

and ΔAIC are presented.

Cobitis magnostriata Cobitis minamorii oumiensis Male Female Male Female

k AIC ΔAIC* k AIC ΔAIC* k AIC ΔAIC* k AIC ΔAIC*

4 917.36 - 2 289.99 - 3 143.00 - 6 253.99 -

3 918.19 0.83 3 292.88 2.89 4 145.11 2.11 5 260.94 6.95 5 923.70 6.34 1 295.47 5.48 5 145.76 2.76 4 262.51 8.52 6 925.49 8.13 4 296.69 6.70 6 150.67 7.67 7 267.25 13.26 * ΔAIC: difference in AIC compared to the best model (with the least AIC).

Results of model selection show that models with k Table 2 Estimated mean of body length of each cohort in the best models (see Table 1) for both sexes of C. (component number) = 4 and k = 2 were selected respectively magnostriata and C. minamorii oumiensis. Cohorts for males and females of C. magnostriata (Table 1). Models (inferred components) are listed in ascending order. with k = 3 and k = 6 were selected respectively for males and females of C. minamorii oumiensis. In any combination of Mean body length (mm) species and sex, models with k = 1, which supports the C. minamorii Cohorts C. magnostriata monomodal distribution, ranked third or lower. The mean value oumiensis of each cohort in the best models was estimated (Table 2). The Male Female Male Female mean values of 70.1 mm and 73.3 mm in males of C. 1st 64.1 68.6 48.5 55.2 magnostriata were estimated in a narrow range. 2nd 70.9 93.3 62.4 63.9

3rd 73.3 - 72.1 72.0 Discussion 4th 79.9 - - 80.7

In this study, the numbers of age classes in spawning 5th - - - 88.1 populations of C. magnostriata and C. minamorii oumiensis 6th - - - 97.5 were estimated from their size–frequency distribution. A noteworthy result is that the number of age classes was estimated as six. It was larger in females of C. minamorii

- 138 - Age class structure of spawning populations of two endangered endemic striated spined loaches in Lake Biwa river system oumiensis than for other species and sexes. The sample size for failed to spawn. Therefore they iteratively ran to the spawning C. minamorii oumiensis females was rather smaller than others, site. This iteration of the spawning failure and running to the but it is unlikely that this largest number of age classes was spawning site in the subsequent season probably caused the attributable to the estimation error. This inference is supported highly multimodal distribution of size frequency distribution of by the ΔAIC: the difference from the smallest AIC among females. A study of the closest related subspecies, Cobitis compared models. In C. minamorii oumiensis females, the minamorii minamorii, has revealed that this species generally ΔAIC of the second best fitting model was largest among other lives two years (Saitoh, 2005). Saitoh (2005) also reported that species/sexes, suggesting that estimation in C. minamorii they lived longer and became larger in body length to retry to oumiensis females was more certain among them. In addition, spawn in the subsequent season if they fail to spawn the first models with k ≥ 6 ranked fourth or lower in model selections, year. More importantly, Saitoh (2005) reported that the local although the model with k = 6 was the best fitting, as described populations often went to extinction after larger individuals above. These results support that the number of age classes of became abundant in the spawning population. In addition, the the spawning population is distinctively larger for C. body length of C. minamorii oumiensis presented in this study minamorii oumiensis females than for other species and sexes is larger than that reported by Nakajima (2012). All of these of this spawning site. facts support that the second hypothesis is likely to be correct: Here, before further discussion, we discuss the validity of C. minamorii oumiensis females in the study site have these estimations. There are apparently some unconvincing iteratively failed at spawning for many years. Actually, earlier inferences drawn from our analyses. First, the two most reports have described that most C. minamorii oumiensis mutually proximal components were found around 70.1 mm females failed to spawn and that gravid females fell from the and 73.3 mm in C. magnostriata males. Second, only two study site to the Lake Biwa at the end of the spawning season components were found for C. magnostriata females, although (Morii et al., 2018a). the body lengths exhibit the largest range found in this study. Females of C. magnostriata show a bimodal distribution of These unconvincing inferences are probably ascribable to the body length. It has already been suggested that C. growth rate variation among individuals. The variance of body magnostriata females spawn for several years (Kawanabe et al., length must increase as individuals grow larger if there such 2005). Nevertheless, the fact that the number of age classes inter-individual variation exists. This increased variation is was less in C. magnostriata females than that of C. minamorii expected to disturb statistical inference. However, we consider oumiensis females reflects that most C. magnostriata females that the main tendency is consistent irrespective of such a successfully spawned, but they died after the second or disturbance; the top three model selections are occupied by the subsequent spawning. These facts suggest that the population sequential number of k (number of components). This result of C. magnostriata is stable now. supports that the estimations are robust to some degree. As explained above, these study results suggest strongly that Moreover, these estimations suggest that the interindividual females of C. minamorii oumiensis have iteratively failed to varieties in growth were greater in C. magnostriata than in C. spawn, causing extraordinary aging in the study site. This minamorii minamorii. spawning failure and extraordinary aging are likely to affect This distinctively larger number of age classes in females of the population of this species adversely. However, this C. minamorii oumiensis probably reflects the spawning habits situation means that the population includes a sufficient of this species over the last few years. Two hypotheses can number of large females, which are valuable for the explain such numerous age classes of spawning females. The conservation of this species. Generally in fishes, juveniles that first hypothesis is that C. minamorii oumiensis females spawn originate from older females that have experienced spawning multiple times during their lives (iteroparity). Some evidence often exhibit better growth and survival than those from young for this species and closely related species, however, suggests or virgin female fishes (Yoneda, 2017). At this study site, room this hypothesis as improbable. An earlier report of the literature exists for the drastic recovery of the C. minamorii oumiensis (Minamori, 1956) suggested that this species was formerly population because many females are running to the site every semelparous; the spawning population exhibited a monomodal year. Therefore, it is anticipated as a key point for the size distribution, implying that they die soon after spawning. A successful conservation of C. minamorii oumiensis to elucidate second hypothesis is that females of this species frequently factors that inhibit females of this species from spawning.

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Finally, we discuss possible factors that have inhibited C. minamorii oumiensis females from spawning at this site. The References study site was constructed in 2005 (Nakano et al., 2015). However, the oldest females of C. minamorii oumiensis Ariga, N., K. Morita, T. Suzuki, N. Sato, M. Okamoto and K. captured in this study were six years old; the number of the Ohkuma (2014) Evaluation of population viability of wild cohort (six) plus a year of the birth. These facts present two chum salmon Oncorhynchus keta in the Toyohira River, important implications about the recent life history of this Sapporo metropolitan watershed, Japan. Bull. Japan. Soc. species. First, the survival rate has been sufficiently high to Sci. Fish 80: 946–955. (in Japanese) allow C. minamorii oumiensis females to live up to seven years. Carlson, M. S., T. P. Quinn and A. P. Hendry (2011) This high rate implies that the spawning-inhibiting factor is Eco-evolutionary dynamics in Pacific salmon. Heredity independent of factors affecting the survival, such as 106: 438–447. nutritional condition or predation risk. Second, the spawning of Gröning, J. and A. Hochkirch (2008) Reproductive interference this species has been inhibited through these eight years between animal species. Q. Rev. Biol. 83: 257–282. (cohort number, 6, plus two years of our survey) at least. This Frost, W. E. (1945) The age and growth of eels (Anguilla inhibition suggests that the spawning-inhibiting factor is anguilla) from the Windermere catchment area. J. Anim. intrinsic to this study site. It is only one spawning site of this Ecol. 14: 106–124. species so far as known at the present, but its situation differs Janko, K., M. Flajšhans, L. Choleva, J. Bohlen, V. Šlechtová, from sites used in the past: C. minamorii oumiensis and C. M. Rábová, Z. Lajbner, V. Šlechta, P. Ivanova, I. magnostriata spawn sympatrically at present but they had Dobrovolov, M. Culling, H. Persat, J. Kotusz and P. Ráb allopatrically spawned in the past; the former and the latter had (2007) Diversity of European spined loaches (genus used temporal and permanent water areas, respectively (Saitoh Cobitis L.): an update of the geographic distribution of the and Matsuda 1990). These facts lead to the hypothesis that Cobitis taenia hybrid complex with a description of new sympatrical spawning with the relative species, C. molecular tools for species and hybrid determination. J. magnostriata, inhibits the successful spawning of C. minamorii Fish. Biol. 71: 387–408. oumiensis. In other words, reproductive interference (Gröning Kawanabe, H., N. Mizuno and K. Hosoya (2005) Freshwater and Hochkirch, 2008; Kuno, 1992) might be occurring between fishes of Japan. pp. 387, Yama-kei Publishers, Tokyo. (in these two species. Some reports of interspecific hybrids among Japanese). Cobitis spp. (Janko et al., 2007; Saitoh, 1990) give evidence Kuno, E. (1992) Competitive exclusion through reproductive that sexual interaction among relative species is possible in the interference. Res. Popul. Ecol. 34: 275–284. genus. In addition, our earlier work at the study site (Morii et Minamori, S. (1955) Local races of spinous loaches. Bull. al., 2018a) demonstrated that C. minamorii oumiensis Biogeogr. Soc. Japan 16–19: 278–282. reproduced less in the year when C. magnostriata adults were Minamori, S. (1956) Physiological isolation in Cobitidae. IV. abundant. Consequently, all existing circumstantial evidence Speciation of two sympatric races of Lake Biwa of the supports the reproductive interference hypothesis, although striated spinous loach. Jpn. J. Zool. 12: 89–104. more direct evidence should be collected. For more rigorous Ministry of the Environment (2015) Red data book 2014. – verification, we should combine some methods, such as threatened wildlife of Japan – Volume 4, Pisces-Brackish behavior observations and controlled experiments in the field. and fresh water fishes. Gyosei Corporation, Tokyo. (in Japanese) Acknowledgments Morii, K., M. Nakano and K. Takakura (2018a) Does simultaneous and sympatric reproduction between two We deeply appreciate help in support of field surveys from A. native spined loaches lead to reproductive interference Honma, D. Kitano, T. Hisaoka, R. Kanai, Y. Nishihira, A. and local extinction? Environ. Biol. Fish. 101: Okada, K. Anan, and T. Anan. We also thank K. Uehara for his 1407–1416. management of the study site and for his assistance with Morii, K., M. Nakano, T. Nishida and K. Takakura (2018b) consensus formation among neighboring farmers. This study Growth patterns of juveniles observed at a shared was partly supported by the Takara Harmonist Fund (in 2016) spawning site in two closely related species of spined and JSPS KAKENHI Grant Number KB17K07273. loaches endemic to the Lake Biwa riverine system;

- 140 - Age class structure of spawning populations of two endangered endemic striated spined loaches in Lake Biwa river system

Cobitis magnostriata and C. minamorii oumiensis. Jpn. J. Saitoh, K. (2005) Cobitis striata species complex. In “The Environ. Entomol. Zool. 29: 49–55. present and future of endangered freshwater fishes: Nakajima, J. (2012) Taxonomic study of the Cobitis striata scenario of fruitful conservation,” (Katano, K. and S, complex (Cypriniformes, Cobitidae) in Japan. Zootaxa Mori, eds), pp. 186–192, Shinzansha, Tokyo (in 3586: 103–130. Japanese). Nakajima, J. (2017) Loaches of Japan. Yama-kei Publishers, Saitoh, K. and H. Aizawa (1987) Local differentiation within Tokyo (in Japanese). the striated spined loach (the striata type of Cobitis taenia Nakano, M., K. Uehara and M. Urabe (2015) Habitat and Complex). Jpn. J. Ichthyol. 34: 334–345. breeding season of striated spined loaches on the Saitoh, K. and S. Matsuda (1990) Spined loaches distributed in northwest coast of Lake Biwa. Jpn. J. Conserv. Ecol. 20: the Lake Biwa water system. The Annual Reports of the 49–58 (in Japanese). Biwako Bunkakan 8: 19–23 (in Japanese). Saitoh, K. (1984) Local races of striated spined loach. Iden Yoneda, M. (2017) Key to fluctuation of the stock biomass of (Heredity) 38: 31–37 (in Japanese). chub mackerel Scomber japonicus: temperature and Saitoh, K. (1990) Reproductive and habitat isolation between maternal effects on early life history traits. Bull. Japan. two populations of the striated spined loach. Environ. Biol. Soc. Sci. Fish. 85: 837(in Japanese). Fish. 28: 237–248. Watanabe, K., H. Ichiyanagi, T. Abe and A. Iwata (2014) Saitoh, K. (1993) Smaller race and the large race of the striated Population viability analysis for the endangered loach spined loach are threatened. Jpn. J. Ichthyol. 40: 394–397 Parabotia curtus in the Lake Biwa – Yodo River system, (in Japanese). central Japan. J. Ichthyol. 61: 69–83 (in Japanese).

琵琶湖固有スジシマドジョウ類 2 種における繁殖個体群の年級群推定

森井清仁 1)* ・高倉耕一 2)

1) 滋賀県立大学大学院環境科学研究科 2) 滋賀県立大学環境科学部

生物の野外個体群の年級群構成についての知見は，その個体群動態を理解する上で必須の知見であり，絶滅危惧 種の効率的な保全に貢献すると考えられる．ともに琵琶湖固有種であるオオガタスジシマドジョウ（オオガタ）と ビワコガタスジシマドジョウ（コガタ）は，分布を狭めており，絶滅が危惧されている．本研究では，滋賀県高島 市の両種の繁殖地において 2015 年から 2017 年に，それぞれの体長について調べ，繁殖個体群の年級群数 k を推 定した．調査の結果，オオガタが 601 個体（オス 488：メス 113）とコガタが 148 個体（60：88）採捕された． オオガタについて，オスでｋ = 4，メスでｋ = 2 と推定された．コガタについては，オスで ｋ = 3，メスで k = 6 と推定された．コガタのメスにおいて年級群数が最大だったことは，本種および近縁種での知見と合わせると，本 種メスがたびたび産卵に失敗し繁殖地への遡上を繰り返していることを示すと考えられた．

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