Research Article |
Corresponding author: Zhao-Liang Guo ( zlguo@fosu.edu.cn ) Academic editor: Saskia Brix
© 2020 Da-Jian Xu, Deng-Xu Li, Xiao-Zhuang Zheng, Zhao-Liang Guo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Xu D-J, Li D-X, Zheng X-Z, Guo Z-L (2020) Caridina sinanensis, a new species of stygobiotic atyid shrimp (Decapoda, Caridea, Atyidae) from a karst cave in the Guizhou Province, southwestern China. ZooKeys 1008: 17-35. https://doi.org/10.3897/zookeys.1008.54190
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From a biodiversity survey of a subterranean habitat near Sinan County, Guizhou Province, southwestern China, a new atyid shrimp of the genus Caridina H. Milne Edwards, 1837, C. sinanensis sp. nov. was discovered. The new species can be separated from other congeners based on a combination of characters including depigmentation in body and reduction of eyes, small pigment spot at the centre of the cornea, the shape of rostrum and the endopod of the 1st male pleopod, and the relatively longer appendix interna on the appendix masculina of the 2nd pleopod. Mitochondrial COI and 16S rRNA gene sequences also support the establishment of the new species. Information on the habitat, and the levels of threat are discussed to guide the conservation of C. sinanensis sp. nov.
Caridina, COI and 16S rRNA, freshwater biodiversity, karst landform, southwestern China, spelaeology, taxonomy
China’s karst landforms, accounting for about a third of the territory, are one of the largest and most spectacular karst sceneries in world (
During an inventory to evaluate the status of a variety of cave fauna along karst cave systems of Guizhou province, southwestern China in 2019–2020, specimens of atyid shrimp were collected from a cave river near Tangtou Town, Sinan County. After detailed examination of these specimens based on a combination of morphological and molecular features (COI and 16S rRNA), we are confident that our specimens have sufficient differences from known species to be recognized as a new species, Caridina sinanensis sp. nov. The present work provides detailed description, illustrations, molecular evidence, standardized diagnoses, colour photographs, and habitat information, as well as a distribution map. In addition, the conservation significance of this species is also briefly described. This raises the total number of subterranean atyid shrimp species known to date from China to 24 species.
The cave where the specimens were sampled is located in Pengjiaao, Tangtou Town, Sinan County, Guizhou Province, southwestern, China, at 27°44'10"N, 108°11'58"E (Fig.
Geological coordinates and basic hydrological and physicochemical parameters of the cave (width, depth, temperature, humidity, pH, CO, CO2, O2, H2S, and dissolved oxygen) were measured with the following instruments: Bosch GLM-30 Laser rangefinder, eTrex Venture GPS locator, JWSA2-2 temperature and hygrometer, LB-MS4X portable gas detector, BDO820 portable dissolved oxygen meter, and BPH-220 pH meter. The environmental physicochemical parameters of the cave are shown in Table
We visited the cave three times, in January and March 2019 and February 2020. Each time some dozens of specimens were observed, and altogether 40 specimens were collected. Samples were collected with a sturdy, long-handled, fine-meshed dip net (mesh size 0.6 mm) and with the aid of headlamps. The sampling procedure was recorded with photographs and video-recordings. Specimens were placed in oxygenated polythene bags, anaesthetized with ice, and transported to the hotel, to be photographed, and fixed in 95% ethanol. Ethanol was changed after 24 h with fresh 75% ethanol.
Specimens were examined using a dissecting microscope (Olympus SZX7). Illustrations and morphometric measurement of selected characters were recorded using a digital camera (DP22) mounted on a stereomicroscope (Olympus SZX7) and Olympus CellSens Entry v. 1.18 software.
The following abbreviations are used throughout the text: alt (altitude), cl (carapace length, measured from the postorbital margin to the posterior margin of the carapace), rl (rostral length, measured from the rostral tip to the postorbital margin), and tl (total length, measured from the rostral tip to the posterior margin of the telson). All measurements are in millimeters.
Specimens were deposited in the Department of Animal Science, School of Life Science and Engineering, Foshan University (FU), Guangdong, China.
The abdominal muscle of the specimens were used for DNA extraction with an EasyPure Genomic DNA Kit (TransGen Biotech, Beijing, China) and then stored in a -20 °C freezer. To construct the molecular phylogeny of selected Caridina species, two mitochondrial gene fragments, 710 bp of the cytochrome oxidase subunit I (COI) and approximately 560 bp of the large ribosomal subunit (16S) were amplified and sequenced on an Applied Biosystems 3730 Analyzer (Applied Biosystems, Foster City, CA, USA) using COI primers LCO1490 and HCO2198 (
Species used in the molecular analysis, with details on sampling locations, GenBank accession numbers (COI, 16S rRNA).
Species | Sampling locality | GenBank accession numbers | |
---|---|---|---|
COI | 16S rRNA | ||
C. breviata | China, from type locality | KP168788 | KP168718 |
China, from type locality | KP168789 | KP168719 | |
C. cantonensis | Qingyuan, China | KP168802 | KP168720 |
Qingyuan, China | KP168803 | KP168721 | |
C. huananensis | Yingde, Qingyuan | MN701607 | MT446452 |
Yingde, Qingyuan | MN701608 | MT446453 | |
C. lanceifrons | Dongfang, Hainan | MN701605 | MT446450 |
Dongfang, Hainan | MN701606 | MT446451 | |
C. mariae | Nankun Mountain, Huizhou | MN701601 | MT446456 |
Nankun Mountain, Huizhou | MN701602 | MT446457 | |
C. nanaoensis | China | KP168792 | KP168755 |
C. serrata | Dong’ao Island, Zhuhai | MN701595 | MT446454 |
Dong’ao Island, Zhuhai | MN701596 | MT446455 | |
C. sinanensis | Sinan Guizhou | MT433962 | MT434873 |
Sinan Guizhou | MT433963 | MT434874 | |
Sinan Guizhou | MT433964 | MT434875 | |
C. trifasciata | Zhuhai China | KP168795 | KP168765 |
Zhuhai China | KP168796 | KP168766 | |
C. zhujiangensis | Dong’ao Island, Zhuhai | MN701603 | MT446448 |
Dong’ao Island, Zhuhai | MN701604 | MT446449 | |
N. palmata | Yangshan, Qingyuan | MN701611 | – |
Yangshan, Qingyuan | MN701611 | – | |
China | – | KP168779 | |
Hong Kong, China | – | KP168780 |
As there are no Caridina longshan and C. alu sequences on the GenBank, and no C. longshan and C. alu specimens were collected, only 22 sequences in this paper were analyzed. It is mainly based on nine species of Caridina shrimp from China, so as to conduct molecular analysis with C. sinanensis sp. nov. All sequences were aligned with MAFFT v. 7.037 software using the auto strategy and normal alignment mode (
Family Atyidae De Haan, 1849
Holotype : Adult male (FU, 2019-01-25-01), tl 16.7 mm, cl 4.8 mm, rl 1.5 mm; a cave river at Pengjiaao, Tangtou Town, Sinan County, Guizhou Province, southwestern, China (27°44'10"N, 108°11'58"E, alt. 294.7 m), 25 Jan. 2019. Paratypes: 1 male (FU, 2019-01-25-02) cl 5.4 mm; 1 male (FU, 2019-01-25-03) cl 6.8 mm; 1 male (FU, 2019-01-25-04) cl 4.8 mm; 2 males (FU, 2019-01-25-05), cl 4.2–6.2 mm; 20 females (9 ovigerous) (FU, 2019-01-25-05), cl 4.9–6.6 mm, sampled together with the holotype.
Caridina semiblepsia Guo, Choy & Gui, 1996. Adult male (FU, 1994-05-17-01), tl 17.5 mm, cl 4.5 mm, rl 0.7 mm; a cave river at Tongpatong, Baojing County, Hunan Province, China, 17 May 1994. Paratypes: 4 males (FU, 1994-05-17-02) cl 4.8–5.6 mm; 5 females (2 ovigerous) (FU, 1994-05-17-03), cl 4.7–6.3 mm, sampled together with the holotype.
Caridina ablepsia Guo & Jiang, 1992. Adult male (FU, 1989-05-23-01), tl 26.8 mm, cl 6.5 mm, rl 1.8 mm; a cave river at Xiaolongtong, Yunshun County, Hunan Province,China, 23 May 1989. Paratypes: 5 males (FU, 1989-05-23-02) cl 5.4–6.7 mm; 6 females (FU, 1989-05-23-03), cl 5.7–6.9 mm, sampled together with the holotype.
Rostrum short, slightly sloping downwards, usually reaching to the end of the 2nd segment, occasionally reaching to the end of the 1st segment or the end of the 3rd segment of antennular peduncle, rostral formula 4–10+10–16/3–11. 1st pereiopod carpus 0.77–0.83× as long as chela, 1.6–1.7× as long as high; chela 1.9–2.2× as long as broad; fingers 1.2–1.3× as long as palm. 2nd pereiopod carpus 1.2–1.3× as long as chela, 4.7–6.1× as long as high; chela 2.2–2.9× as long as broad; fingers 1.6–2.3× as long as palm. 3rd pereiopod propodus 3.8–4.1× as long as dactylus, with 9–11 thin spines on the posterior and lateral margins. 5th pereiopod propodus 3.7–4.1 × as long as dactylus, with 11–13 thin spines on the posterior and lateral margins, dactylus terminating in one claw, with 38–44 spinules on flexor margin. Endopod of male 1st pleopod extending to 0.45–0.50× exopod length, distal half usually curved posteriorly in the natural, occasionally not bent backwards, wider proximally, subrectangular, 2.4–2.7× as long as wide, appendix interna well developed, arising from distal 1/3 of endopod, reaching beyond end of endopod. Appendix masculina of male 2nd pleopod rod-shaped, reaching to 0.51 length of endopod, appendix interna reaching to 0.93 length of appendix masculina. Uropodal diaeresis with 10–12 movable spinules. Eggs size (without eyespots) 0.67–0.82 × 1.29–1.38 mm, eggs size (containing embryos with eyes) 0.98–1.02 × 1.16–1.47 mm.
Body
(Fig.
Rostrum
(Fig.
Eyes
(Fig.
Carapace
(Fig.
Antennule
(Fig.
Caridina sinanensis sp. nov. A, B carapace and cephalic appendages, lateral view C eye D antennule E antenna F mandible G maxillula H maxilla I first maxilliped J second maxilliped K third maxilliped A, C holotype (FU, 2019-01-25-01) D–K paratype (FU, 2019-01-25-02) B paratype (FU, 2019-01-25-03).
Antenna
(Fig.
Mandible
(Fig.
Maxillula
(Fig.
Maxilla
(Fig.
First maxilliped
(Fig.
Second maxilliped
(Fig.
Third maxilliped
(Fig.
First pereiopod
(Fig.
Second pereiopod
(Fig.
Third pereiopod
(Fig.
Caridina sinanensis sp. nov. A first pereiopod B second pereiopod C third pereiopod D dactylus of third pereiopod E fourth pereiopod F fifth pereiopod G dactylus of fifth pereiopod H–J first pleopod K second pleopod L telson M diaeresis of uropodal exopod N spermatophore A–G, I–N paratype (FU, 2019-01-25-02) H paratype (FU, 2019-01-25-04).
Fourth pereiopod
(Fig.
Fifth pereiopod
(Fig.
First four pereiopods with epipod. Branchial formula typical for genus.
First pleopod
(Fig.
Second pleopod
(Fig.
Telson
(Fig.
Female carrying a number of 20–32 eggs, sized eggs 0.67–0.82 × 1.29–1.38 mm (without eyespots), and 0.98–1.02 × 1.16–1.47 mm (with eyespots).
Colouration (Fig.
Caridina sinanensis is named after Sinan County, where the type locality is located.
Six Caridina species lacking body pigmentation and having a small black spot on each eye are known from Chinese subterranean aquatic habitats: C. acuta, C. alu, C. demenica, C. longshan, C. semiblesia, and C. sinanensis These taxa can be readily separated into two groups by the rostrum shape and indentation. In the first group including C. acuta, C. demenica, and C. semiblesia, the rostrums are similarly lanceolate and short, with fewer teeth or unarmed. In the second group including C. alu, C. longshan, and C. sinanensis , the rostrums are long, reaching at least to the end of the 2nd antennular segment, mostly beyond the end of scaphocerite, and armed with dorsal and ventral teeth. Caridina. sinanensis is morphologically close to C. longshan in sharing a similar spination pattern, the anterior region of endopod on the 1st male pleopod folded backwards, and the variably pigmented cornea. Caridina. sinanensis can be distinguished from C. longshan by the relatively longer appendix interna on the appendix masculina of the 2nd pleopod (reaching about 0.93 of appendix masculina vs 0.80 in C. longshan), the length of 6th abdominal segment distinctly longer than the telson (vs same length of telson in C. longshan), and telson with posteromedian projection and lack of spinules on the surface of posterior telsonic spines (caudal spines) (vs lacking posteromedian projection and possessing spinules in C. longshan). Caridina. sinanensis can be easily separated from C. alu by its short rostrum (reaching to the end of the 2nd antennular peduncle vs reaching to the end of scaphocerite in C. alu), the carpus of 1st and 2nd pereiopods are slender (length to breadth ratio 1.6–1.7 and 4.7–6.1 versus 1.3 and 2.6 in C. alu), the telson with posteromedian projection (vs lacking in C. alu), and male with completely different shape of the endopod of 1st pleopods and appendix masculina of the 2nd pleopods. Caridina. sinanensis sp. nov. also shows close similarity with C. semiblesia in the ratios of various segments of the 1st and 2nd pereiopods, and the shape of endopod of the 1st pleopod in males. In addition to a longer rostrum that slopes downwards, C. sinanensis also differs from C. semiblesia in having the end of the palp of the 1st maxilliped being broadly rounded and without a finger-like tip (vs ending in a finger-like tip in C. semiblesia), the smaller eggs (0.98–1.02 × 1.16–1.47 mm vs 1.05–1.15 × 1.37–1.71 mm), and the stout and long appendix interna of the appendix masculina on the 2nd pleopod (appendix interna almost same size as appendix masculina and reaching about 0.93 of appendix masculina vs distinctly slender and reaching about 0.80 in C. semiblesia).
Including the GenBank sequences, we analysed 22 COI sequences and 22 16S rRNA sequences in total. The new sequencing results are corrected for 621~bp (COI) and 487~bp (16S) for subsequent analysis. Three specimens of Caridina sinanensis were used in the molecular phylogenetic analysis shown in Figures
Caridina sinanensis, sp. nov. lives in an aphotic subterranean waterbody where the source of energy may come from allochthonous materials carried or washed into the cave, as there are particulates of vegetable debris in the water. Based on our observation on the shrimp’s feeding behavior and intestinal contents, this species feeds on detritus and microorganisms from the bottom sediments with its brush-tipped chelae and mouthparts, and the full intestine suggests that the foods are relatively abundant (Fig.
Leeches co-occurred with atyid shrimp in the subterranean waters, camel crickets were common on the cave rocks, especially in the dark zone, and a blind unpigmented species of millipede was found crawling along the rocks in the dark zone (Fig.
In general, the populations of other cave-dwelling species were very small, while shrimp were moderately abundant. Competition for food and habitat seems insignificant, possibly because the groundwater was enriched with particulate organic matter and predators (such as leeches) were not abundant. Leeches are the natural enemies of the shrimp; they attach to the carapace, branchial chambers and appendages where they feed on the hemolymph of the shrimps. Parasitism certainly confers negative impact on populations of the new species, but accurate population data on the shrimp are lacking.
The sex ratio and reproduction season were preliminary inferred based on three sampling times. On 25 January 2019, 18 individuals were collected, including two adult males, 10 adult females (six ovigerous), and six subadult females. The sex ratio (male to female) is 1:8, and the percentage of ovigerous females is 60%. On 18 March 2019, 18 individuals were caught, including five adult males, 10 adult females (three ovigerous), and three subadult females. The sex ratio is 1:2.6, and the percentage of ovigerous females is 30%. In 18 February 2020, four individuals were caught including one adult male and three adult females (one ovigerous). The sex ratio is 1:3, and the percentage of ovigerous females is 33%. These results showed that the number of males was significantly less than females in the population from January to March. The causes responsible for the skewed sex ratio in favor of females may worth further study.
The ovigerous females comprised 60%, 33%, and 30% of mature females, respectively, in populations from January to March. One male carrying a spermatophore on the intermediate of the fifth walking legs was observed from specimens collected in January (Fig.
Females carried 20–32 eggs, size of undeveloped eggs (without eyespots) were 0.67–0.82 × 1.29–1.38 mm, size of developed eggs (containing embryos with eyes) 0.98–1.02 × 1.16–1.47 mm. The females of this species carry a small number of large eggs and produce eggs with a large amount of yolk and reduced number of larval stages. It is believed that abbreviated larval development may occurs in this species, larval direct development into benthic hatchlings that resemble miniature adults.
We are trying to understand embryonic development and hatching of this species. On 20 March 2019, five ovigerous females were transported to the laboratory for rearing, but unfortunately, after 7 days, the shrimps died.
Cave ecosystems are an invaluable resource, providing an ideal refuge for cave-dwelling species. Cave shrimp communities are particularly vulnerable to human disturbance, particularly groundwater pollution due to the local agricultural activities (fertilization, herbicide, and pesticide) and overexploitation (domestic usage and agricultural irrigation). These appear to be responsible for the pollution and degradation of subterranean habitat, but the extent of the impact is a little known. If groundwater become contaminated, local aquatic organisms certainly are at risk. Maintaining healthy groundwater shrimp communities requires the reduction of anthropogenic impacts, such as minimizing the use of agricultural pesticides, herbicides, and fertilizers by local farmers. It is suggested that the local government should ration the use of groundwater resources.
The Announcement of the Ministry of Agriculture and Rural Areas of China (CITES Appendix aquatic wild species of China, no. 69, 2018), fails to list freshwater shrimps in the CITES threatened categories. Since Caridina sinanensis is a new species, no conservation status has been assigned. According to the criteria listed in the IUCN Red List categories (
Environmental parameters | Unit | Light zone | Twilight zone | Dark zone | ||||||
---|---|---|---|---|---|---|---|---|---|---|
2019.1.25 | 2019.3.18 | 2020.2.15 | 2019.1.25 | 2019.3.18 | 2020.2.15 | 2019.1.25 | 2019.3.18 | 2020.2.15 | ||
Temperature(air) | °C | 9.2 | 15.5 | 9.8 | 9.8 | 17.7 | 14.2 | 14.8 | 18.4 | 15.1 |
Temperature(water) | °C | / | / | / | 20.1 | 20.2 | 20.2 | 20.2 | 20.3 | 20.2 |
Humidity(air) | % | 76 | 78 | 80 | 90 | 92 | 93 | 94 | 96 | 97 |
pH(water) | / | / | / | 6.6 | 6.5 | 6.7 | 6.6 | 6.6 | 6.7 | |
oxygen(air) | % | 28 | 27 | 29 | 23 | 25 | 24 | 21 | 22 | 22 |
Hydrogen sulfide(air) | mg/kg | 0.45 | 0.48 | 0.44 | 0.37 | 0.40 | 0.39 | 0.21 | 0.25 | 0.22 |
Carbon monoxide(air) | mg/kg | 17.0 | 17.2 | 17.2 | 16.9 | 16.1 | 14.9 | 11.5 | 12.2 | 11.7 |
Carbon dioxide(air) | mg/kg | 270 | 273 | 280 | 270 | 274 | 283 | 355 | 348 | 367 |
Dissolved oxygen(water) | mg/L | / | / | / | 8.3 | 8.0 | 8.7 | 8.3 | 8.0 | 8.7 |
Caridina sinanensis sp. nov. is not blind, as the eyes still contain considerable pigmentation, although we found one male specimen lacking pigments in its cornea (Fig.
The freshwater atyid species associated with caves are relatively numerous in China. The number of subterranean atyids recorded now totals 24, but only 13 species can be regarded as real troglobionts. Of these, five have reached the advanced stage in which the faceted cornea of the eyes is absent, namely: Caridina alba, C. ablepsia, C. caverna, Mancicaris sinensis, and Typhlocaridina lanceifrons. Eight species with reduced eyes but presence of pigmented cornea are: C. acuta, C. alu, C. demenica, C. longshan, C. semiblesia, T. liui, T. semityhplata, and C. sinanensis sp. nov. In order to contribute to the management of the vulnerable subterranean ecosystems and their highly specialized endemic stygofauna, there is an urgent need to accelerate scientific research, including but not limited to collecting the much-needed information on taxonomy, life history, ecology, and distribution, and to design monitoring programs of subterranean species, especially for those that are under the most intense threats from anthropogenic factors.
As molecular analysis has become a crucial step towards resolving taxonomic problems, delimitations, and the real biodiversity of atyids, a combined morphological and molecular data is necessary for the description of a new taxa. Ideally, this process will involve to comprehensive information on color, physiology, and ecology. In Chinese karst landforms, as many caves are explored and the molecular identification applied, the species list will increase with future surveys.
This study was supported by the Special Fund for Central Public Welfare Research Institutes (grant no. PM-zx097-201904-134), the Running Cost for Key Laboratory of Utilization and Breeding of Aquatic Resources in Tropical and Subtropical Areas, Ministry of Agriculture and Rural Affairs (grant no. 9020190008) and the Investigation on Crustaceans in Priority Area of Mangrove Diversity Protection in Guangxi Zhuang Autonomous Region (grant no. kh19051). Thanks are also due to Dr Kayan Ma (Sun Yat-sen University) for modifying the manuscript. We acknowledge the subject editor Saskia Brix, copy editor Robert Forsyth, and three reviewers (Yixiong Cai, Thomas Iliffe, and one anonymous reviewer) for providing their valuable suggestions, which greatly improved the manuscript.