FECUNDITY AND REPRODUCTIVE STRATEGY OF PTYCHOBARBUS DIPOGON POPULATIONS FROM THE MIDDLE REACHES OF THE YARLUNG ZANGBO RIVER

LIU Hai-Ping， LIU Yan-Chao， ， LIU Shu-Yun， SONG Xiao-Guang， ， TSERING Luo-Jie， LIU Meng-Jun，LIU Le-Le and RAO Chang-Wei

(1. Institute of Fisheries Science， Tibet Academy of Agricultural and Animal Husbandry Sciences， Lhasa 850002， China; 2. Food Science College， Tibet Agriculture and Animal Husbandry University， Nyingchi 860000， China; 3. Tanggu Township， Linzhou County， Lhasa 850000， China; 4. Azha Township， Zhanang County， Shannan 856000， China; 5. Research Institute of Animal Husbandry and Veterinary Medicine， Tibet Academy of Agricultural and Animal Husbandry Sciences， Lhasa 850002， China;6. Tibet Animal Epidemic Disease Prevention and Control Center， Lhasa 850000， China; 7. Pangduo Township， Linzhou County， Lhasa 850000， China)

Abstract: The Ptychobarbus dipogon of the Schizothoracinae subfamily， is an endemic economic fish species in Tibet Autonomous Region， China. Due to environment deterioration and invasion of alien species the population size of this species is decreasing; a study is urgently required to determine fecundity and reproductive strategy in order to conserve this natural resource. In this study， to investigate the fecundity and its relationship of body length， weight and age， we captured 1030 individuals in the middle Yarlung Zangbo River during two separate periods， one from February to March in 2013 and the other from February to June in 2014. The results showed that the standard length (SL) of males is concentrated in the 325—400 mm range，and the SL of females is above 375 mm. Sixty five females were at stages IV and V of sexual maturity， with SL 320—500 mm， weight 411.6—1328.0 g. Using the SL 50% method， the first female sexual maturity age was estimated to be 13.0 years and about 360.90 mm， while the first male maturity age was estimated to be 13.5 years and length 354.53 mm. The distributions of egg sizes， gonadal developmental stages and gonadosomatic indexes show that this species follows a synchronous spawning pattern concentrated in the period February to March， with an absolute fecundity at 3487 eggs， with a fecundity to SL ratio of 7.2 eggs/mm and fecundity to weight of 4.3 eggs/g. The absolute fecundity is positively correlated to the SL and the weight， but not significantly correlated to age. The overall male-female ratio was 1.23∶1. Suggestions for its conservation have been made based on its low fecundity， late maturity and short breeding period.

Key words: Tibet Autonomous Region; Ptychobarbus dipogon; Fecundity; Reproductive strategy

ThePtychobarbus dipogonRegan is a member of the genusPtychobarbusin the subfamily Schizothoracinae. It is a fish species with specialized adaptation for the middle reaches of the Yarlung Zangbo River[1]. Many sources have reported on its fecundity，including general-purpose surveys of local fish fau na[2]， with some specialized studies on wide-ranging topics， such as the species nutritional value[3]， feeding organs and behaviors[4]， reproductive strategies[5]，chromosome diversity[6]， age， growth and population dynamics[7]， correlation between age， growth and mortality[8]， age determination[9]， chromosome count[10]， length-weight relationship[11]， and mitochondrial sequencing[12].

Presently， the most in-depth works onP. dipogontend to focus on the evolution of Schizothoracine in the context of the rise of the Tibetan Plateau and subsequent climate changes. Cao，et al. offered a systematic postulation that the evolution of Schizothoracines based on fish trait variations is connected to environmental changes[13]. Through molecular phylogenetics and biogeography of the specialized Schizothoracine fishes， He，et al. demonstrated that their origin is intrinsically connected to the stepwise rise of the Tibetan Plateau; their evolution and distribution are a direct result of adaptation to those changing environments[14]. Wang investigated the molecular phylogenetics of East Asian Cyprinds and found that their earliest branching event may be concurrent with the development of the East Asian Monsoon climate caused by the plateau rise[15].Yang，et al.[16]analyzed the mitochondrial DNA of Schizothoracines， and concluded their evolution was likely connected to the plateau rise. Estimated historical altitudes using fish fossils suggest a correlation between the rising height of the plateau and the evolution of Schizothoracines[17].Transitional Cyprinid fossils of the Tertiary period，found in northern Tibet Autonomous Region， proved the eventual emergence of Schizothoracinaes to be an adaptation to the cooling climate[18]. The current Tibetan Schizothoracines are still in a process of speciation， with numerous taxonomic traits yet to be stabilized， suggesting that rapid environmental changes are still occurring in the plateau ecosystems[19].

In recent years， the mainstream and branches of the Yarlung Zangbo River have suffered ecological problems including reduced wildlife populations， decrease of individual fish size， invasive alien species[2]and habitat deterioration[1]. Due to the low temperatures of highland lakes and rivers， Tibetan fish species naturally grow[20]， sexually mature and reproduce at a slower rate compared to fish elsewhere[19，21]. Currently， the deteriorating aquatic ecosystems endanger the habitats， feeding and breeding of Tibetan Schizothoracine fish[22]， also， the dam projects may obstruct their migratory passages and further effect their activities of foraging， overwintering and spawning[23]. The continued existence ofP. dipogonis under direct threat， and any such disturbance in the system may damage its population in ways that may be impossible to recover， or take a long time to recover from[24].

To rationally conserve and manage the wild resources ofP. dipogon， it is imperative to study the biology of reproduction and reproductive strategies of this species. In this paper， we studied the sex ratio，fecundity， reproductive period， age and size at first sexual maturity， and reproductive strategies. Our findings provide valuable information for the rational conservation and exploitation ofP. dipogon.

1 Materials and Methods

1.1 Fish sample collection

We captured 19 female individuals ofP. dipogonat stages IV and V from February to March in 2013 in the Xigaze segment of the Yarlung Zangbo River in Xaitongmoin County (altitude ～4000 m， denoted by A1)， and collected more individuals from February to June in 2014 in the upper reaches of the Lhasa River， a tributary of the Yarlung Zangbo River(altitude ～3800 m， A2)， of which 33 individuals were caught in June， 37 in February， and more than 130 in each of the remaining months. In total， 1030 individuals were collected. Specimens were sampled using floating gillnets (mesh size 7.5 cm) and bottom gillnets (mesh size 6.5 cm). All fish were transported by live fish transport vehicles to the Tibetan Fish Reproduction & Nursery Base at the School of Tibet Agriculture and Animal Husbandry University for research. Their standard length， weight， gutted weight，ovary weight and egg size were measured with standard length (SL) accurate to within 1 mm， and weight to within 0.1 g. Six vertebrae were taken from each fish as materials to determine fish age.

1.2 Preparation and observation of vertebral columns

For each individual， its vertebral column was separated from muscular attachments， briefly immersed in boiling water and then the remaining muscle and other tissue cleaned by brush. After drying， scissors were used to carefully remove all ribs and protrusions around the column. The column was then numbered and preserved. A stereo microscope was used to observe the column and determine age[25].

1.3 Sex ratio

Individuals were divided into groups by standard length at intervals of 50 mm for study of the sex ratios ofP. dipogonat different growth stages[26]. The chi-squared test is used to determine if the sex ratio conforms to 1∶1.

1.4 Breeding season

The breeding season ofP. dipogonwas determined by comparing the distribution of gonadal developmental stages and egg sizes， and variation of the gonadosomatic index (GSI) among different months. Specifically， the breeding season is characterized by the appearance of stage IV and V gonads in substantial numbers.

Fat (K) andGSIwere calculated using the following functions:

Where:Wis body weight，W0is gutted weight，W1is gonad weight，Kis fat， andGSIis the gonadosomatic index.

1.5 Egg sizes and distribution

The six-stage gonadal rank criteria developed by the Yellow Sea Fisheries Research Institute was used to determine the developmental stage of gonad via visual inspection[27]. The gonadal ranks were denoted with Roman numerals from Ⅰ to Ⅵ， egg sizes were measured by diameter using matured or near-matured ovaries at stages Ⅳ and Ⅴ[28]. The distributions of egg sizes in different months were used to determine the spawning pattern.

1.6 Size and age at first sexual maturity

Male and female individuals were respectively divided into intervals of 10 mm， and theSL50%(the length at which an individual has a 50% probability to be sexually mature) method was used to determine sampleSLand age at first sexual maturity.P=1/{1+exp[–k(SLTmid–SL50)]}[26]. Where:P: percentage of mature individuals with length in the intervalSL.k: the slope.SLTmid: median of the length interval.SL50: average standard length at first sexual maturity.The age at first sexual maturity (A50) was appraised by fitting the logistic function to the proportion (P) of mature fish:P=1/{1+exp[–k(A–A50)]}[26]. where:A:the age of the individuals，A50: the age at first sexual maturity.

1.7 Fecundity

A number of female fish at stages Ⅳ and Ⅴwere randomly chosen to measure their total ovary weight and weight of sampled eggs (accurate to 0.1 g).The sampled eggs were preserved in a 10% Formalin solution. The number of sampled eggs was counted，and the absolute and relative fecundities were calculated as follows:

Absolute fecundity = (number of sampled eggs/weight of sampled eggs) × ovary weight

Fecundity relative to weight = absolute fecundity/gutted weight (g)

Fecundity relative to length = absolute fecundity/length (mm)

1.8 Data analysis

Excel 2007 and SPSS 21 were used for data processing and regression analysis of fecundity and distribution of egg sizes. One-way analysis of variance(ANOVA) and Tukey test were used to detect significant differences inGSIandKper month. A difference was regarded as significant atP＜0.05.

2 Results and Analyses

2.1 Secondary sexual characteristics

In non-breeding seasons， female and maleP.dipogonare indistinguishable in appearance. During the breeding seasons， patches of lighter colors can be seen on the back， caudal peduncle， and fin rays of the mature male fish， especially near the anal fin; these patches are coarse to the touch. The mature female fish has no such patches， but its abdomen becomes swollen and soft， while its cloaca protrudes and turns red.

2.2 Sex ratio

A total of 1030 individuals were acquired. The sex of 68 individuals could not be determined. 530 males and 432 females were identifiable， and the male-female ratio was 1.23∶1， which significantly differed from 1∶1 by Chi-squared test (χ2=4.99，P＜0.05). The standard length of individuals were in the range 155—550 mm， with weight in the range 46.5—1704.5 g. TheSLof most males was concentrated in the range 325—400 mm， and theSLof most females was above 375 mm (Fig. 1). Analysis shows 350 mm to be a dividing point. The male-to-female ratio increases with length below 350 mm， and decreases with length above. No male specimens longer than 500 mm were caught (Fig. 2).

2.3 Distribution of gonadal developmental stages over months

Tab. 1 illustrates features ofP. dipogonat different gonadal stages. Females in stage Ⅱ were seen throughout February to June; stage Ⅲ appeared in February， March and May; stage Ⅳ appeared from February to May and were most prominent in February (45%); stage Ⅴ were seen from February to April， and reached to a peak of 36.43% in May; stageⅥ appeared from April to June (Fig. 3A). Male individuals in stage Ⅳ were more prominent from February to June， and stage Ⅴ appeared from February to April(Fig. 3B). It can be seen that the breeding season of the species lasts from February to April， with March as the height of breeding activities.

2.4 Variations of gonadosomatic index and fatness in different seasons

The females caught from the Xaitongmoin area had remarkably higherGSIvalues than the females from the upper Lhasa River in the same months of 2014. Among the 2014 specimens， theGSIis highest in March， and not very different from April to June.There is a significant difference between theGSIof female specimens from Xaitongmoin in March and all female specimens from the Lhasa River (P＜0.05); the difference is insignificant between the months from February to May in the Lhasa River specimens， but the femaleGSIfrom these months are significantly different from June (P＜0.05) (Tab. 2).

Fig. 1 Body length distribution of P. dipogon

Fig. 2 Sex ratios of P. dipogon in different SL groups

Tab. 1 Characteristics of gonadal development in P. dipogon

The females show higher average fatness than the males. Among females from the upper Lhasa River， there was significant difference between their fatness in June and that in the other months (P＜0.05);significant differences in fatness also exist in males between February， June， and the months in-between(P＜0.05). This is an indication of the continual maturation of gonads from February to April (Tab. 3).

Fig. 3 Breakdown of gonadal developmental stages for female (A) and male (B) P. dipogon

Tab. 2 Female GSI values of P. dipogon between sampling areas and periods

Tab. 3 Variations of male/female fatness for P. dipogon for different months

2.5 Fecundity

The eggs from 60 female individuals at stages IV and V were counted， withSLin the 320—500 mm range， and weight in the 507.0—1566.0 g range. The absolute fecundity ranged from 1078 to 9590 (3487±1731)， the relative fecundity toSLfrom 3.2 to 13.9(7.2±2.5) eggs/mm， and relative fecundity to weight from 1.6 to 7.6 (4.3±1.4) eggs/g. For absolute fecundity， another 106 individuals were added to the calculation (Tab. 4).

The data displays a general trend of absolute fecundity increase withSL(Fig. 4A)， with the best fit equation:F= 11.4SL–1717.3，R2= 0.139，n=60(P＜0.01); absolute fecundity also tends towards increase with weight (Fig. 4B)， with the best fit equation:F= 4.5W–360.7，R2= 0.313，n=166 (P＜0.01).There is no significant correlation between absolute fecundity with age (P＞0.05) (Fig. 4C).

2.6 Egg sizes

In total， 7749 eggs at stages IV and V were measured， obtaining an average size of (3.63±0.25) mm.Female specimens caught in both the Xaitongmoin and Lhasa River areas displayed an egg size peak around 3.6 mm in both February and March. For females in the Lhasa River， the April peak was around 4.2 mm， while some eggs were as small as 2.3 mm，indicating they were approaching the end of breeding season in April. A2-March has the highest peak. Except for A2-April， most plots are single-peaked (Fig. 5).

2.7 Size at first sexual maturity and smallest sexually mature individual

Logistic regressions to the proportion (P) of mature fish were performed on the size (SL50) and age(A50) at 50% maturity of females and males as follows:

Size at 50 % maturity:

Female:P= 1/{1 + exp [–0.042 (SL–360.900)]}，R2= 0.937

Male:P= 1/{1 + exp [–0.030 (SL–354.530)]}，R2= 0.780

Age at 50 % maturity:

Female:P= 1/{1 + exp [–0.252 (A–13)]}，R2=0.829

Male:P= 1/{1 + exp [–0.099 (A–13.5)]}，R2=0.461

At first sexual maturity， the femaleP. dipogonhas aSLof 360.90 mm， 13.0 years old， the male has aSLof 354.53 mm， 13.5 years old by fitting the logistic regressions. Among the 1030 captured individuals，the smallest female individual has aSLof 338.0 mm，weighs of 429.5 g， 18 years old; the smallest male has aSLof 310.0 mm， weighs of 327.0 g， 9 years old.

2.8 Living environment and reproductive habits

The caught specimens show thatP. dipogontends to inhabit the middle reaches of the Yarlung Zangbo River at an altitude of 3580—4000 m. Larger individuals tend to inhabit medium to deep layers of the river， in areas with strong currents， deep water，and riverbed of large rocks. Smaller individuals tend to live in shallower areas with wide river surfaces，slow water flow， and gravel riverbed. The species largely consume benthic animals and phytoplanktons.From mid-February to early April， the species migrate to shallow areas with wide and calm surfaces，abundant plankton with more gravel and less mud in the riverbed， suitable for breeding purposes.

Tab. 4 Fecundity of P. dipogon

Fig. 4 Correlations between absolute fecundity and SL， weight and age in P. dipogon

Fig. 5 Egg size distribution of P. dipogon in different months

3 Discussion

3.1 First sexual maturity

In recent years， many studies[29—37]paid attention to the reproductive biology of Schizothoracine fishes. Wu[22]noted that Schizothoracines from different regions exhibit different spawning patterns related to their climate and environment， also the sex ratios of their reproductive populations vary with time，SLgroup， and habitat[38].

The age of first sexual maturity is a determining factor for specie reproductive potential via the influences of the duration of its fertility and the size of reproductive populations[39]. Fish population age and size at sexual maturity are the combined result of its bionomic and genetic features and environmental factors; fishing pressure， abundance of predators and feed and composition of the population are some examples of biological and non-biological factors[40]. Food supply and other factors that affect growth rate all have an impact on first sexual maturity[29]. The majority of Schizothoracines become sexually mature in the third to sixth year， and the males tend to mature earlier than the females[22，30，33，35，37]; but there are also species that require ten years or longer to mature[32].

Among the Schizothoracines of Tibet Autonomous Region (Tab. 5)， the first sexual maturity ofP.dipogoncomes the second latest， only earlier thanSchizothorax oconnori. The first maturity age in our study is consistent with Li’s[5]finding. Compared to overall age， there is a stronger correlation between first sexual maturity age andSL. For female and maleP. dipogon， theSLat first sexual maturity are 360.90 mm and 354.53 mm respectively， at ages of respectively 13.0 and 13.5. Their slow growth rate and late maturity should be directly connected to the scarcity of food and low temperature in the harsh highland environment. This conclusion is similar to that proposed forSchizothorax waltoni[29]，Oxygymnocypris stewartii[31]，Schizothorax oconnori[32]，Gymnocypris przewalskii[35]andSchizopygopsis malacanthus baoxingensis[37]. TheSL50and first sexual maturity ages ofP. dipogonare a good basis to devise measures for its monitoring and management.

3.2 Individual fecundity and reproductive strategy

FecundityFecundity is an important characteristic in showing how a species or population reacts to environmental change， and its understanding can help predict population changes. The fecundity of fish is influenced by a multitude of factors including environment， such as water quality and other fish populations in the habitat， and the population itself. We find individuals of similar age carry different numbers of eggs， which may suggest differences in individual nutrition intake caused by the scarcity of food[32]. Different groups in the same area also have different breeding periods and significantly different fecundities[28].

Most Schizothoracines have yellow demersal eggs of 1.54—4.0 mm diameter. Their absolute fecundities ranged from 2300 to 16000， and their average relative fecundities ranged from 10 to 45 eggs per gram[35，37]. Generally speaking， Schizothoracine absolute fecundity increases with length and weight[28].

The absolute fecundity ofP. dipogonis positively correlated to length and weight， and is not significantly related to age， this is similar to that reported forSchizothorax oconnori[32]， andOxygymnocypris stewartii[31]，Schizopygopsis malacanthus baoxingensis[37].

Tab. 5 Comparison of first sexual maturity ages among cold-water Schizothoracine fishes

Compared to the absolute fecundity of other cold-water Schizothoracine (Tab. 6)， theP. dipogonis less fecund， yet has larger eggs. This strategy ensures relatively high hatchability and survivability by giving eggs and larvae a good endogenous nutritional supply， and makes them more likely to achieve their first feeding as fry. This is consistent with the conclusion from a prior study ofSchizothorax oconnori[32].

Tab. 6 Comparison of absolute fecundities of cold-water Schizothoracine fish

With an absolute fecundity of 3330.6， theP.dipogonranks fairly low among currently studied Tibetan Schizothoracines. Comparison to Li’s[5]result shows a decline in the fecundity ofP. dipogonin the same area， suggesting an increasing threat to its propagation in the future. The difference may be attributed to the long-term overfishing of the species.

Reproductive strategiesEach fish population has evolved its own reproductive strategy to ensure the survival of its offspring， which can include the season and duration of breeding activities[40]. The spawning of fish is dictated by a combination of internal reproductive cycle and external signals (e.g.temperature， daylight duration and water flow)[28]， of which water temperature and daylight duration may be the most crucial[55，56]. TheP. dipogonhas a short breeding season that concentrates breeding activities in March， and ends in early April. Because eggs take longer to hatch in the cooler waters of the plateau， an early spawning date may ensure a better environment for the early development of the larvae and fry[37].

The fatness of a fish indicates its nutritional and environmental condition[28]. Females of theP. dipogonhave higher fatness than males. Their fatness shows a slight increase in the early part of the breeding season from the end of winter to early spring; it decreases after breeding due to the energy expended by spawning or spermiation; the fatness slightly increases again for a while after breeding.

TheP. dipogonis a cold-water fish native to the Tibetan environment， with low fecundity， late sexual maturity， and short breeding period. These characteristics make them highly sensitive to environmental damage and， once reduced， their repopulation would be difficult even over a long period. This fact promotes the importance of protecting their habitat and regulation of fishing.

3.3 Spawning pattern

Knowledge of spawn pattern is essential to the estimation of fish species fecundity， and for tracing its repopulation and bionomic strategy. Based on the developmental morphology of oocytes in ovaries， Shi[57]divided spawning patterns into three categories: completely synchronized， batch-synchronized， and batchunsynchronized.

Spawning patterns can be determined using histological observation of gonads， cyclical variation ofGSI， and frequency distribution of egg sizes[37]. TheP.dipogononly exhibits one egg size peak in March， and two peaks in April， which proves the existence of a completely synchronized pattern， similar toOxygymnocypris stewartii，[31]Schizothorax waltoni[29]，Schizothorax oconnori[32]， andSchizopygopsis stoliczkae[34].

3.4 Suggestions on conservation

The construction of new hydropower dams[58]，reservoirs[59]， mines， quarries[60]， and other projects has increasing impacted the habitats of theP. dipogon， including spawning grounds， feeding， overwintering and migration. In light of its low fecundity， late sexual maturity and short breeding season， we have the following suggestions for policymakers regarding its preservation:

(1) Stricter regulation and control should be rigorously enforced on overfishing， unlicensed quarrying of stone and sand， and industrial wastewater and solid wastes in order to protect the species habitat.

(2) Measures should be taken to address the impact on fish migration routes of closed-off water bodies， such as reservoirs.

(3) The breeding season ofP. dipogoncould be designated a closed season， to protect its reproductive population， eggs and larvae.

(4) Release of alien fish species should be prohibited， as they tend to out-compete local species.Regulation should be imposed on the Buddhist animal release rituals popular among tourists， also outreach efforts should be made to inform the populace of the necessity to preserve local ecosystems.