Jpn. J. Environ. Entomol. Zool. 29（2）： 49－55（2018） 環動昆 第 29 巻 第 2 号：49－55（2018） Original Article

Growth patterns of juveniles observed at a shared spawning site in two closely related of spined loaches endemic to the Lake Biwa riverine system; magnostriata and C. minamorii oumiensis

Kiyohito Morii1)*, Mitsunori Nakano2), Takayoshi Nishida3) and Koh-Ichi Takakura3)

1) Graduate School of Environmental Science, The University of Shiga Prefecture, 2500, Hassaka-cho, Hikone-City, Shiga 522-8533, 2) Bioresouces and Environmental Sciences, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi-shi, Ishikawa-ken, 921-8836 Japan 3) Environmental Science, The University of Shiga Prefecture, 2500, Hassaka-cho, Hikone-City, Shiga 522-8533, Japan

（Received: March 26, 2018；Accepted: May 10, 2018）

Abstract Two spined loach , Cobitis magnostriata Nakajima, 2012 and C. minamorii oumiensis Nakajima, 2012 are endemic to the Lake Biwa riverine system. Although they were once distributed throughout the coasts of Lake Biwa, the distributions of both species have narrowed up to the present day: they have become regarded as endangered species. For three years (2015–2017), we conducted field surveys at a ditch in Takashima, Shiga Prefecture and recorded the growth patterns of juveniles of both species. A series of surveys captured 700 and 40 juveniles, respectively, of C. magnostriata and C. minamorii oumiensis. The growth of juveniles of C. magnostriata was linear until early June, after which it reached a plateau. That saturation occurred at 20–25 mm body length. Few individuals exceeded 25 mm. Results suggest that the saturation was the result of sufficiently grown juveniles (20 mm or larger) running down to Lake Biwa. In 2015, the level at saturation for C. magnostriata was higher. The density of conspecific juveniles was thought to be lower than in other years. These results suggest that intra-specific resource competition affected this species. We were unable to trace the growth of C. minamorii oumiensis juveniles except in 2015 because ever fewer juveniles were captured in 2016 and 2017: our surveys captured fewer juveniles of this species decreased year by year. The study site is apparently the only spawning site for C. minamorii oumiensis at present. These facts support that C. minamorii oumiensis faces an immediate crisis. Overcoming this crisis requires elucidation of details of the C. minamorii oumiensis biology and hastening of the development of effective conservation tactics.

Keywords: Cobitis striata species complex, conservation, fallow field biotope, life history, resource competition, spawning

Introduction anecdotal reports have been published recently (e.g. Kawamura et al., 2015; Nakano et al., 2015). In recent years, many freshwater fishes in Japan have Both Cobitis magnostriata Nakajima, 2012 and C. become endangered. On the national red list in Japan (Japanese minamorii oumiensis Nakajima, 2012 are endemic species Ministry of Environment, 2014), 123 species are listed as distributed in the Lake Biwa riverine system. They are critically endangered (CR) or endangered (EN). An example is mutually similar in morphology (Saitoh, 1984), but C. the Cobitis striata species complex of 5 species and 11 magnostriata is larger than C. minamorii oumiensis in body subspecies occurring in Japan (Nakajima, 2017). All are listed length and egg size (Minamori, 1956). Saitoh and Matsuda as CR, EN or Vulnerable (VU) in the red list. Conservation of (1990) reported that adult individuals of C. magnostriata these species/subspecies is an urgent task. Details of the inhabit Lake Biwa during most of their life, except during the biology of this species complex, which is necessary for spawning season. During May–June, they migrate to and effective conservation, are little known, although some spawn at permanent water areas surrounding the lake, such as

＊Corresponding author: [email protected]

- 49 - Morii et al ditches and side streams. Saitoh and Matsuda (1990) also reported that adults of C. minamorii oumiensis inhabit ditches connecting to Lake Biwa and the lake. They run to and spawn at temporary water areas such as paddy fields during June–July. A recent report (Nakano et al., 2015) has suggested that both species spawn sympatrically in a site, which is the only site where C. minamorii oumiensis is certain to have spawned in recent years. Some reports of the relevant literature (Saitoh, 1993; Saitoh and Matsuda, 1990) have suggested that C. magnostriata and C. minamorii oumiensis are distributed throughout the coasts of Lake Biwa. The distributions of both species have narrowed to their present state, however. Therefore, both species have been Fig. 1 Study site on June 12, 2016. The water in the ditch designated as CR in the Red Data Book of Shiga Prefecture flows from the bottom right to the upper side in the (Scientific Committee for Research into the Wildlife in Shiga picture. Prefecture, 2016), and as EN in the national Red Data Book 30–200 cm wide, ca. 15 cm deep at most parts and ca. 30 cm (Japanese Ministry of the Environment, 2015). Particularly, C. minamorii oumiensis has been limited to the northern part of deep at the deepest part. Neither the ditch bottom nor wall has the western coast of the Lake Biwa (Saitoh, 1993). These facts been paved. Phragmites australis (Cav.) Trin. Ex Steud occurs urge the conservation of both species. Nevertheless, the in places. The water origin of the outer canal was pumped-up groundwater. migrating from the outer canal to the study biology of these species is as scarce as that of other members site run through the fish way installed at the ditch outlet, of of Cobitis striata species complex. Knowledge of ecological which the drop was ca. 6 cm. characteristics of juveniles is fundamentally important for their effective conservation. Nevertheless, not even descriptive The water level of the site is fundamentally dependent on studies of juveniles have been reported for the two species, the schedule of rice cultivation at the paddy fields located probably because they could not be identified morphologically. across the outer canal: the water level remained high during May and June. In 2015, for example, the water intake to the For this study, we strove to obtain knowledge that can ditch began on May 8. Water was drained on July 4. In this contribute to their conservation. We conducted field surveys at season, the water level of the outer canal was elevated. Then the spawning site described above and applied genetic markers to identify collected juveniles. Then, we elucidated details the water flowed into the ditch of the study site. In 2016 and related to the growth of juveniles of both species. 2017, however, we manipulated the downstream weir to change the schedule. In 2016, the water intake began on May 10. It was drained once on July 2, as in previous years. Three Materials and Methods days later (on July 5), however, the water level was elevated

again. The condition continued until July 29. The water level Study site We conducted a series of surveys at a ditch in Shin-Asahi, management in 2017 was similar to that in 2016. The water Takashima, Shiga Prefecture, Japan. At this site, adults of both level was high during April 22 through June 30 and during July C. magnostriata and C. minamorii oumiensis were found 6 through 23. To maintain the water level, we used an engine pump to pump water up to the site from the external canal (Nakano et al., 2015). This site, originally a fallow paddy field 2 during the two durations of high water level in 2016 and 2017. (total area of ca. 3000 m ), was reconstructed into the Therefore, the ditch did not dry up even during the dry grassland where a ditch ran in 2005 (Fig. 1). This site was intervals. constructed with the aim of the water clarification by settling suspended particles in drainage water from paddy fields and the restoration of temporary water area used by fishes for Field survey We surveyed the growth of juveniles of the two species at spawning. This study site is connected to Lake Biwa through the study site. Generally in ichthyological studies, the juvenile the outer canal of approximately 360 m. Nakano et al. (2015) stage is defined as the development stage with fin rays and described the site details. The ditch is ca. 230 m in total length,

- 50 - Growth patterns of juveniles of juveniles in two endemic spined loaches, sharing a spawning site scales. Our field surveys captured larvae that were smaller than juveniles, but the larvae were few, as described below. In this M 1 2 3 4 5 6 7 8 study, we do not discriminate juveniles and larvae. We refer to them as juveniles for simplicity. These surveys were conducted while the water level at the site was high: during May 9 through July 4 in 2015, May 10 – July 29 in 2016, and April 22 2000 bp – July 23 in 2017. We conducted surveys once a week, 9 times 1000 bp in all, in 2015. In 2016 and 2017, however, we conducted 500 bp surveys once every two weeks during May–June and once a week during the other period, 9 and 10 times in all. We 100 bp conducted a total of 55 hand-nettings on each day of the surveys. The points of hand-netting were scattered so that they Fig. 2 Results of species identification of juveniles both C. homogeneously cover the entire ditch. Hand-netting was done magnostriata and C. minamorii oumiensis by PCR. The lanes of 1–3 and 4–8, respectively, show C. minamorii by one person wading within the ditch using a hand-net (38 × oumiensis and C. magnostriata. The lane M shows a 38 cm, mesh size of 1 mm). The hand-net was dragged on the ladder marker (Gene Ladder 100; NIPPON GENE Co., ditch bottom along ca. 1 m length lines that were arranged Ltd., Tokyo). arbitrarily for each survey. Juveniles were not identifiable based on their morphology. Therefore, we used genetic Table 1 Number of captured individuals of C. magnostriata markers for identification. Juveniles were stored in 99% and C. minamorii oumiensis from 2015 until 2017 Number of juveniles Species ethanol. Their standard body length was measured using a captured each year stainless steel ruler (H-101A; Shinwa Rules Co., Ltd., Sanjo) at 2015 2016 2017 the laboratory at The University of Shiga Prefecture. The 99% C. magnostriata 178 245 277 ethanol specimens were stored at about 4°C until subsequent analysis. C. mimamorii oumiensis 26 13 1

Identification of juveniles followed by 35 cycles at 98°C for 10 s, 57°C for 30 s, and We used a molecular biological method to identify juveniles 72°C for 60 s; with final extension at 72°C for 4 min. After because it is impossible to discriminate species based on their PCR was completed, 5 μl out of 15 μl of the obtained PCR morphology. This method was used for all juvenile specimens. product was electrophoresed on a 1.5% agarose gel and stained Extraction of DNA from the specimens described above was using ethidium bromide. We discriminated each sample to performed using a kit (Easy DNA Extraction Kit Version 2; either of two species based on the amplified fragment length. Kaneka Corp., Osaka) according to the attached protocol. Using the extracted DNA solution as a template, PCR was Results performed with 15 μl per reaction according to the attached protocol (Emerald Amp PCR Master Mix; Takara Bio Inc., In a series of surveys conducted over three years, 700 and 40 Kusatsu). Multiplex PCR was performed using primers juveniles of C. magnostriata and C. minamorii oumiensis were designed based on the nucleotide sequence data of the captured, respectively. All juveniles that we captured were ND4-ND5 region of mitochondrial DNA in reference to Saitoh identified using specific PCR primer sets (Fig. 2). The number et al. (2010). The species were identified according to the of juveniles of C. magnostriata increased year by year. By result. Forward primers were 5'-gcccttcatcttatcccagtta-3 'and contrast, that of C. minamorii oumiensis decreased (Table 1). 5'-attattttgcttagccaacaccat-3', which were suitable, respectively, The body length of juveniles of C. magnostriata grew for C. magnostriata and C. minamorii oumiensis, using linearly from May until early June. The growth gradually 5'-ctcggtgagtaaggggttttta-3' as the common reverse primer. saturated after mid-June (Fig. 3). The saturation levels Using this primer set, amplified products of 140 bp and 477 bp, differed among years. Growth was ca. 20 mm in 2015, but ca. respectively, are obtainable for template DNA derived from C. 15 mm in later years. In 2017 only, short individuals appeared magnostriata and C. minamorii oumiensis. The PCR was in mid-July. In that year, juveniles were found in early and performed under the following conditions: 95 °C for 5 min; mid-May, although they were found in late May or later in

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Fig. 3 Increase of the body length (mm) of C. magnostriata Fig. 4 Increase of the body length (mm) of C. minamorii juveniles from May until July. The top, middle, and oumiensis juveniles from May until July. See legends of bottom panels present the growth in 2015, 2016 and 2017, Fig. 3 for a detailed explanation. respectively. The open and filled arrows show the days of the inflow and drainage of water to/from the study site. 2015 and 2016. The day of inflow of April 22, 2017 is not shown here Similarly, the body length of juveniles of C. minamorii because it is out of the panel. Round symbols denote outliers. Bars represent the maximum and minimum oumiensis grew linearly until early June and then more slowly values within 1.5 times the width of the box from lower after mid-June (Fig. 4). However, we were unable to trace the or upper quartile. The heavy line in the box represents the growth in 2016 and 2017 because few juveniles were captured median. in these years.

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Discussion adult fish from immigrating to the study site in 2016. However, no such drop in water level was found at the second water For this study, we conducted a series of surveys for three inflow in 2017. Adults of C. magnostriata were observed to years to clarify the growth of juveniles of both C. magnostriata immigrate to the study site. These facts suggest that the second and C. minamorii oumiensis at the study site, where two spawning in C. magnostriata can be artificially enhanced by species spawn sympatrically. Based on the results, we adequate manipulation of the water level, which could described their growth patterns and annual differences. contribute to the efficient conservation of C. magnostriata. The growth patterns of juveniles of C. magnostriata were We were able to trace the growth of C. minamorii oumiensis almost the same in the three years: their growth was linear juveniles only in 2015 because fewer C. minamorii oumiensis until early June; thereafter, it saturated. The saturation juveniles were captured in this study than C. magnostriata. occurred at 20–25 mm body length. Few individuals exceeded Therefore, we can not deeply discuss the life history, including 25 mm. It is more reasonable to infer that the saturation the growth of juveniles of C. minamorii oumiensis here. The showed that the sufficiently grown juveniles (20 mm or larger) only tendency demonstrated in this study is that the body ran down to Lake Biwa, not that the juveniles’ growth ceased. length of juveniles of C. minamorii oumiensis was slightly less This idea is supported by the known biology of C. than that of C. magnostriata, similarly to adults of the two magnostriata: it inhabits Lake Biwa except for the spawning species. season (Saitoh and Matsuda, 1990). Nakano et al. (2015) Another notable tendency was found in the relation between reported that juveniles of the two species were not collected in the body length and the abundance of in C. magnostriata the outer canal connected to the study site. This fact strongly juveniles. The number of captured juveniles in this study suggests that the sufficiently grown juveniles of C. probably reflected their relative density because we magnostriata did not remain in the outer canal, but instead ran hand-netted them in a consistent manner. Then the actual directly to Lake Biwa. The biology of juveniles of C. density of C. magnostriata juveniles was thought to be lower magnostriata has remained unclear. This study revealed that in 2015. On the other hand, the juveniles had greater body the juveniles of C. magnostriata grow in the spawning site for length in 2015 than in the other years. These facts suggest that more than a month after hatching and that they run down to the larger body length of juveniles in 2015 can be ascribed to Lake Biwa when they exceed 20–25 mm in body length. the density effect in the juvenile stage. In other words, Two notable annual differences were found in the growth juveniles of this species might have competed for resources, pattern of C. magnostriata. First, the juveniles were found probably zooplankton, as their food, in 2016 and 2017. The earlier in 2017 than in other years, probably because of the consequent food shortage might have slowed their growth irregular schedule of the water level management in 2017. before they reached the threshold size to run down from the Water was allowed to flow into the site approximately two site. Actuary, some reports suggest that a food shortage limited weeks earlier than in previous years. The first juvenile of C. the growth rate in organisms that use temporary water area (e.g. magnostriata in each year was captured consistently at the time Ogawa, 2008) although temporary water areas have been point about two weeks after water inflow, which suggests that generally thought to have an abundance of prey species for C. magnostriata spawn immediately after the water inflow, juvenile fishes (Williams and Coad, 1979). irrespective of the day of water inflow. Second, only in 2017 This larger size of C. magnostriata juveniles in 2015 implies were small juveniles found in July, which suggests that C. also that inter-specific competition between the two spined magnostriata spawned again in July 2017, triggered by the loaches was not severe compared to intra-specific competition. second inflow on July 6. The second water inflow was More juveniles of C. minamorii oumiensis were captured in conducted in 2016 also. The water level was insufficiently 2015 than in any of the prior three years. The C. magnostriata elevated because of the low water level in the outer canal in the juveniles were larger in that year than in other years, as duration. The water level of second water inflow in 2016 was described above. If the inter-specific competition were 15 cm lower than the water level of the inflow in 2017. extremely severe the C. magnostriata juveniles in 2015 would Farmers who managed the surrounding paddy fields removed have become as small as observed in other years. Based on the water weeds before the second water inflow, which enhanced facts presented above, it is reasonable to infer that the water flow in the outer canal and consequently lowered the intra-specific competition is more important for these two water level. This lowered water level might have prevented spined loaches than their mutual inter-specific competition.

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However, many prey species for juvenile fishes exist in Minamori, S. (1956) Physiological isolation in . IV. temporary water areas (Williams and Coad, 1979). Therefore, Speciation of two sympatric races of Lake Biwa of the food resource depletion effects on the growth of C. striated spinous loach. Jpn. J. Zool. 12: 89–104. magnostriata juveniles must be investigated. Ministry of the Environment (2015) Red data book 2014. – This study revealed some characteristics of life histories of Threatened wildlife of Japan – Vo lu me 4 , Pisces – C. magnostriata and C. minamorii oumiensis, especially their Brackish and fresh water fishes. Gyosei Corporation, spawning and the growth of juveniles at a spawning site, Tokyo (in Japanese). although the sample size was rather small in the latter species. Nakajima, J. (2017) Loaches of Japan. Yama-kei Publishers, More detailed data must be collected for C. minamorii Tokyo. oumiensis, but the situation constitutes a crisis. This species is Nakano, M., K. Uehara and M. Urabe (2015) Habitat and now in danger of immediate extinction. Apparently, this study breeding season of striated spined loaches on the site is the only spawning site for C. minamorii oumiensis. northwest coast of Lake Biwa. Jpn. J. Conservation Ecol. Alleviating this crisis requires elucidation of the biology of C. 20: 49–58 (in Japanese with English summary). minamorii oumiensis in greater detail and hastening of the Ogawa, R. (2008) Acheilognathus longipinnis: a symbol fish of development of effective conservation tactics. flood plains with natural hydrometeorological environments. Jpn. J. Ichthyol. 55: 144-148 (in Japanese). Acknowledgments Saitoh, K. (1984) Local races of striated spined loach. Iden (Heredity) 38: 31–37 (in Japanese). We deeply thank A. Honma, D. Kitano, T. Hisaoka, R. Kanai, Saitoh, K. (1993) Smaller race and the large race of the striated Y. Nishihira, A. Okada, K. Anan, and T. Anan for their support spined loach are threatened. Jpn. J. Ichthyol. 40: 394–397 with field surveys. We also thank K. Uehara for his (in Japanese). management of the study site and his help with consensus Saitoh K, W. J. Chen and R. L. Mayden (2010) Extensive formation among neighboring farmers. This study was partly hybridization and tetrapolyploidy in spined loach fish. supported by Takara Harmonist Fund (in 2016) and JSPS Mol. Phylogenet. Evol. 56: 1001–1010. KAKENHI Grant Number KB17K07273. Saitoh, K. and S. Matsuda (1990) Spined loaches distributed in the Lake Biwa water system. The Annual Reports of the References Biwako Bunkakan 8: 19–23 (in Japanese). Scientific Committee for Research into the Wildlife in Shiga Kawamura, T., S. Suzuki, K. Usami, M. Komuro, N. Kawai, T. Prefecture (2016) Red data book of Shiga prefecture 2015. Watabe and H. Yokoi (2015) Habitat of loach (Cobitis Sunrise, Hikone, Japan (in Japanese). minamorii tokaiensis) in the agricultural drainage Channel. Williams, D. D. and B. W. Coad (1979) The ecology of Research Bulletin of the Aichi Agricultural Research temporary streams III. Temporary stream fishes in center 47: 115–118 (in Japanese). Southern Ontario, Canada. Int. Rev. Ges. Hydrobiol. 64: 501-515.

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琵琶湖固有シマドジョウ 2 種（Cobitis magnostriata および C. minamorii oumiensis）における共有繁殖地での稚 魚の成長パターン

森井清仁 1)*・中野光議 2)・西田隆義 3)・高倉耕一 3)

1) 滋賀県立大学大学院環境科学研究科

2) 石川県立大学生物資源環境学部 3) 滋賀県立大学環境科学部

オオガタスジシマドジョウ Cobitis magnostriata とビワコガタスジシマドジョウ C. minamorii oumiensis はとも に琵琶湖固有種である．両種は琵琶湖沿岸域に広く分布していたが ，現在では限られた地域にのみ分布し，絶

滅危惧種として扱われている．本研究では両種が同所的に繁殖する滋賀県高島市の土水路で，2015 年から 2017 年の間に調査を行い，両種の稚魚の成長パターンを記録した．オオガタスジシマドジョウの稚魚は， 5 月から 6 月上旬にかけて急激に成長し，6 月中旬以降に成長が緩やかになった．これは，十分に成長した稚魚（ 20 mm 以上）が調査地から琵琶湖に流下していったためであると考えられた．同種の密度が他年よりも低い 2015 年は 稚魚の成長が他年よりも大きな体長で停滞した．これは種内の資源競争 が稚魚の成長に影響を与えていること を示唆している．ビワコガタスジシマドジョウの稚魚については，本研究で得られたサンプルが少なく，詳細

な成長に関しての議論はできなかった．

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