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1 2010 Homepage: Developed and maintained: ENDEMIC SPIDERS IN CHINA Online at : : lisq@ioz.ac.cn Dr. Shuqiang Li Key Laboratory of Zoological Systematics and Evolution Institute of Zoology Chinese Academy of Sciences 1 Beichen West Road, Chao-Yang Dist. Beijing , PR. China Tel.: (office) Fax : (office) lisq@ioz.ac.cn

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3 FROM LEFT: LIN Linghui, ZHAO Qingyuan, HOU Zhonge, XIAO Yonghong, WANG Chunxia, WANG Xiaoxiao, Gerard van der Velde*, Dirk Platvoet*, LIANG Aiping*, ZHANG Yuanyuan, YANG Lu, GAO Caixia, LI Shuqiang, YAO Zhiyuan * ,,,,,,

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5 Xiao Y.H., Zhang J.X. & Li S.Q Male-specific (Z)-9-tricosene stimulates female mating behaviour in the spider Pholcus beijingensis. Proceedings of the Royal Society B - Biological Sciences 277: [SCI cited] 2. Hou Z.E. & Li S.Q Intraspecific or interspecific variation Delimitation of species boundaries within the genus Gammarus (Crustacea, Amphipoda, Gammaridae), with description of four new species. Zoological Journal of the Linnean Society 160: [SCI cited] 3. Ba J.W., Hou Z.E., Platvoet D., Li Z, Li S.Q Is Gammarus tigrinus (Amphipoda, Crustacea) becoming cosmopolitan through shipping? Predicting its potential invasive range using ecological niche modeling. Hydrobiologia 649(1): [SCI cited] 4. Liu J., Li S.Q. & Pham D.S The coelotine spiders from three national parks in Northern Vietnam (Araneae: Amaurobiidae). Zootaxa 2377: [SCI CITED] 5. Lin Y.C. & Li S.Q Leptonetid spiders from caves of the Yunnan-Guizhou Plateau, China (Araneae: Leptonetidae). Zootaxa 2587: [SCI cited] 6. Tang G. & Li S.Q Crab spiders from Xishuangbanna, Yunnan Province, China (Araneae, Thomisidae). Zootaxa (2703): [SCI CITED] 7. Tang G. & Li S.Q Crab spiders from Hainan Island, China (Araneae, Thomisidae). Zootaxa 2369: [SCI CITED] 8. Yao Z.Y. & Li S.Q Pholcid spiders of the genus Khorata Huber, 2005 (Araneae: Pholcidae) from Guangxi, China. Zootaxa 2594: [SCI cited] 9. Hou Z.E., Pan Y.T. & Li S.Q Description of a new blind species of Heterokamaka (Crustacea: Amphipoda: Kamakidae) from Dianshan Lake, China. Zootaxa 2359: [SCI CITED] 10. Li J.C. & Li S.Q Description of Caridina alba, a new species of blind atyid shrimp from Tenglongdong cave, Hubei Province, China (Decapoda, atyidae). Crustaceana 83(1): [SCI CITED] 11. Lin Y.C. & Li S.Q New armoured spiders of the family Tetrablemmidae from China. Zootaxa 2440: [SCI CITED] 12. Lin Y.C. & Li S.Q Long legged cave spiders (Araneae, Telemidae) from Yunnan Guizhou Plateau, southwestern China. Zootaxa 2445: [SCI CITED] 13. Liu J., Li S.Q. & Jäger P Huntsman spiders (Araneae: Sparassidae) from Xishuangbanna Rainforest, China. Zootaxa 2508: [SCI CITED] 14. Liu J. & Li S.Q New Coelotine spiders from Xishuangbanna Rainforest, Southwestern China (Araneae: Amaurobiidae). Zootaxa 2442: [SCI CITED] 15. Liu J. & Li S.Q. 2010, The Notiocoelotes spiders (Araneae: Agelenidae) from Hainan Island, China. Zootaxa 2561: [SCI CITED] 16. Pan Y.T., Hou Z.E. and Li S.Q Description of a new Macrobrachium species (Crustacea: Decapoda: Caridea: Palaemoidae) from a cave in Guangxi, with a synopsis of the stygobiotic Decapoda in China. Journal of Cave and Karst Studies 72(2): [SCI CITED] 17. Platvoet D., Van der Velde G., & Li S.Q Clothespin setae of Dikerogammarus villosus (Sowinsky, 1894) as possible mediators between environmental gas concentrations and

6 pleopodal beat frequency. AMPIS Report 7. Crustaceana 83 (10) : [SCI CITED] 18. Song Y.J. & Li S.Q The spider genera Araeoncus Simon, 1884 and Diplocephalus Bertkau, 1883 (Araneae, Linyphiidae) of China. Zoosystema 32(1): [SCI CITED] 19. Tong Y.F. & Li S.Q Eight new spider species of the genus Pholcus (Araneae, Pholcidae) from China. Zootaxa 2355: [SCI CITED] 20. Tong Y.F. & Li S.Q The goblin spiders of the genus Opopaea (Araneae, Oonopidae) in Hainan Island, China. Zootaxa 2327: [SCI CITED] 21. Wang C.X. & Li S.Q., 2010, New species of the spider genus Telema (Araneae, Telemidae) from caves in Guangxi, China. Zootaxa 2632: [SCI CITED] 22. Wang C.X. & Li S.Q., 2010, Four new species of the spider genus Telema (Araneae, Telemidae) from Southeast Asia. Zootaxa 2719: [SCI CITED] 23. Wang C.X. & Li S.Q., 2010, Two new species of the spider genus Cataleptoneta from Balkan Peninsula (Araneae, Leptonetidae). Zootaxa 2730: [SCI CITED] 24. Wang X.P., Zhu M.S. & Li S.Q A review of the coelotine genus Eurocoelotes (Araneae: Amaurobiidae). Journal of Arachnology 38: [SCI CITED] 25. Zheng, Y.C., Deng D., Li S.Q, and Fu J Aspects of the breeding biology of the Omei mustache toad (Leptobrachium boringii): ploygamy and paternal care. Amphibia-Reptilia 31: [SCI CITED] 26. Liu J., Li S.Q. & Pham D.S Caponiidae (Araneae), a newly recorded family from Vietnam. Acta Zootaxonomica Sinica ( ) 35(1): Gao C.X. & Li S.Q., Molione lemboda sp. nov., a new spider (Araneae, Theridiidae) from Xishuangbanna of Yunnan, China. Acta Zootaxonomica Sinica ( ) 35(1): Gao C.X. & Li S.Q., One new spider species of the genus Cephalobares from Yunnan, China (Araneae, Theridiidae). Acta Zootaxonomica Sinica ( ) 35(2): Song Y.J. & Li S.Q., Three new record genera and three new species of Erigoninae from China (Araneae, Linyphiidae) Acta Zootaxonomica Sinica ( ) 35(4): Gao C.X. & Li S.Q., Artema atlanta, a pantropical species new for China (Araneae, Pholcodae). Acta Arachnologica Sinica ( ) 19(1): (10): , ( ) ( ),

7 Downloaded from rspb.royalsocietypublishing.org on August 26, 2010 Proc. R. Soc. B (2010) 277, doi: /rspb Published online 12 May 2010 Male-specific (Z)-9-tricosene stimulates female mating behaviour in the spider Pholcus beijingensis Yong-Hong Xiao 1,3, Jian-Xu Zhang 2 and Shu-Qiang Li 1, * 1 Key Laboratory of Zoological Systematics and Evolution, and 2 State Key Laboratory of Integrated Management of Pest Insects and Rodents in Agriculture, Institute of Zoology, Chinese Academy of Sciences, Beijing , People s Republic of China 3 College of Life Sciences, Jinggangshan University, Ji an, Jiangxi , People s Republic of China Chemical signals play an important role in spider sexual communication, yet the chemistry of spider sex pheromones remains poorly understood. Chemical identification of male-produced pheromonemediating sexual behaviour in spiders has also, to our knowledge, not been reported before. This study aimed to examine whether chemically mediated strategies are used by males of the spider Pholcus beijingensis for increasing the probability of copulation. Based on data from gas chromatography mass spectrometry analysis, electroantennography assay and a series of behavioural tests, we verified that (Z)-9-tricosene is a male-specific compound in the spider P. beijingensis. This compound acts as an aphrodisiac: it increases the likelihood that a female will mate. Mate-searching males release (Z)-9-tricosene to stimulate sexual behaviour of conspecific females. In the two-choice assay, however, sexually receptive females show no preference to the chambers containing (Z)-9-tricosene. This indicates that the male pheromone of P. beijingensis is not an attractant per se to the conspecific females. This is, to our knowledge, the first identification of a male-produced aphrodisiac pheromone in spiders. Keywords: (Z)-9-tricosene; male pheromone; aphrodisiac; Araneae; Pholcidae 1. INTRODUCTION Accurate mate identification is an important component in spider sexual communication. The exchange of chemical signals is probably the first type of communication in spiders that serves to bring males and females together (Weygoldt 1977). Although chemical communication has long been recognized in spiders, most of the early research concentrated on behavioural assays of spider sex pheromones (Prenter et al. 1994; Searcy et al. 1999; Anderson & Morse 2001; Kasumovic & Andrade 2004; Roberts & Uetz 2005; Leonard & Morse 2006; Stoltz et al. 2007). The chemistry of spider pheromones remains poorly understood (Gaskett 2007). Some studies have investigated the chemistry of spider pheromones, and a few sex pheromones of females have been identified to date (Schulz & Toft 1993; Prouvost et al. 1999; Papke et al. 2000, 2001; Trabalon et al. 2005; Xiao et al. 2009; Chinta et al. 2010; Jerhot et al. 2010). Although all the identified spider pheromones are released by females and received by males, there are known bioassay examples of male pheromones in spiders that mediate the courtship behaviour of conspecific males or females. For example, silk extracts from mature males of the wolf spider, Schizocosa ocreata, can affect the frequency of agonistic displays and inhibit courtship behaviour among conspecific males. This is indicative of male-produced pheromones bound to the silk acting as a male male inhibitor (Rao Ayyagari & Tietjen 1987). In the funnelweb spider, Agelenopsis aperta, all females enter a * Author for correspondence (lisq@ioz.ac.cn). quiescent state prior to the initiation of mating (Singer et al. 2000). The males can complete mating when the female is physiologically or behaviourally inactive. Becker et al. (2005) demonstrated that the courting males emit an airborne pheromone to induce female quiescence during courtship. Males of a small theridiid spider (Argyrodes sp.) have a protuberance on the front of their heads. This secretes an aphrodisiac that is sucked on by females during mating(legendre & Lopez 1974; Becker et al. 2005). No one, to our knowledge, has reported, however, a chemical identification of male-produced pheromone-mediating sexual behaviours in spiders. Pholcid spiders (Araneae, Pholcidae), known colloquially as daddy-long-leg spiders, are among the dominant web-building spiders distributed worldwide. They occupy a wide variety of habitats ranging from leaf litter to tree canopies; several species occur in caves and in close proximity to humans (Huber 2005). Sexual selection by female choice occurs in pholcids (Uhl 1998; Huber 1999; Uhl et al. 2005). Pholcus beijingensis is a common species found in various caves near Beijing in China. Spiders of this species usually construct untidy webs in dark and damp recesses of cave entrances (Chen & Li 2005). Pholcus beijingensis is polygamous and males and females have multiple mating partners. Males abandon their webs after their last moult to search for potential mates while the females wait on their webs for males (Chen & Li 2005). We have demonstrated that a combination of (E,E)-farnesyl acetate and hexadecyl acetate acts as a female-produced sex pheromone in this species (Xiao et al. 2009). The mate-searching males (MMs) can locate potential mates based on the sex pheromone Received 10 April 2010 Accepted 22 April This journal is q 2010 The Royal Society

8 Downloaded from rspb.royalsocietypublishing.org on August 26, Y.-H. Xiao et al. Male-produced aphrodisiac in a spider associated with the female s silk. The approach of an MM, unlike a female or immature male, rarely triggers predatory or aggressive behaviour in the sexually receptive female (Chen & Li 2005; Xiao et al. 2009). Therefore, we hypothesized that MMs might emit scents acting at a close range as sexual attractants and/or stimulants for the conspecific mature females. To test the hypothesis that a male-produced pheromone exists in P. beijingensis, we isolated and identified chemical signals from the body extract of P. beijingensis and tested for chemoreceptor responses to the compound using electroantennography. We confirmed behavioural responses with female choice tests and behavioural assays. 2. MATERIAL AND METHODS (a) Spiders Adult and subadult specimens of P. beijingensis were collected in March and April of 2008 at the entrance of a cave located to the southwest of Beijing ( N, E). Mean body weights of the adult males, adult females and subadult males (SMs) were mg (n ¼ 10), mg (n ¼ 10) and mg (n ¼ 12), respectively. Each spider was kept in a glass cuvette (4 cm inner diameter (i.d.) 12 cm deep) with a small moistened wad of cotton on the bottom to provide humidity. The cuvettes were put in a climatic chamber (RXZ 268B, Ningbo Jiangnan Instrument Factory) under a 12 L : 12 D photoperiod regime at 258C (day) and 238C (night). About fruitflies (Drosophila melanogaster) were provided to each spider for food once a week. (b) Chemical procedures As the site of pheromone production in the spider is unknown, we extracted the whole body for gas chromatography mass spectrometry (GC MS) analysis. Whole-body extraction was carried out as described (Budenberg et al. 1993; Leal et al. 1994). Sexually receptive females (RFs), MMs and SMs supplied the body extraction samples. In courtship sequences of P. beijingensis, the male unfolding its pedipalps towards the female is seen as a critical behavioural pattern because it implies courting success. At this point, the female turns from passive to active and approaches the male for mating (Xiao et al. 2009). To determine whether the adults were reproductively active, we paired each male with an adult female on her web and checked for courtship behaviour. We removed the male from the web when it unfolded its pedipalps towards the female. Ten spider pairs from the total 15 pairs were found to be sexually receptive in a 1 h observation period. These sexually RFs and males were chosen for the chemical analysis. Each spider was put into a 100 ml cuvette, which was inserted into a 1.5 ml screw-topped vial (Agilent Technologies, Santa Clara, CA, USA). Then, 40 ml dichloromethane (purity greater than 99.5%, Beijing Fine Chemical Company, Ltd, Beijing, China) was put into the cuvette to extract compounds from the spider body. Twenty-four hours later, we removed the spider from the cuvette and stored the remaining solution at 2208C until analysis by GC MS. Analytical GC MS was performed on an Agilent Technologies Network 6890N GC system coupled with a 5973 Mass Selective Detector using the NIST/EPA/NIH Mass Spectral Library (2002 version; Agilent Technologies). Chemstation software (Windows 2000) was used for data acquisition and processing. The GC was equipped with a 30 m HP5 2 MS capillary column (0.25 mm i.d mm film thickness; Agilent Technologies). Helium was used as the carrier gas at a flow rate of 1.0 ml min 21. The temperature of the injector was set at 2808C. Two microlitre aliquots of each sample were injected in the splitless mode. As dichloromethane is very volatile, we measured the volume of the solution remaining from the body extracts after injecting 2 ml into the GC MS instrument so that we could calibrate the proportion of the extract injected for titre analysis. The oven temperature was programmed to increase from 100 to 3008C at58c min 21 and then held for 15 min. Electron-impact ionization used 70 ev and the scanning mass ranged from 30 to 450 amu. Compounds were identified tentatively by matching their gas chromatographic retention times and mass spectra with authentic analogues of the mass spectral library. Synthetic (Z)-9-tricosene (purity greater than 99.4%; Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) and tricosane (purity greater than 99%, Alfa Aesar, a Johnson Matthey Co., MA, USA) were used to confirm the identification of natural products after separation on a non-polar column (HP52MS, 0.25 mm 30 m 0.25 mm) and a polar column (DB 2 wax, 0.25 mm 30 m 0.25 mm, J&W Scientific, Folsom, CA, USA). We determined the content of (Z)-9-tricosene from the body extract of one male P. beijingensis using an external standard. The synthetic compound (Z)-9-tricosene was diluted sequentially in dichloromethane to concentrations of 0.001, 0.01 and 0.1 mg ml 21. We injected 1 ml aliquots of each prepared solution into the GC MS instrument. By comparing peak areas of the body extracts and those of the (Z)-9- tricosene solutions, we estimated that the peak areas of most male body extracts were larger than that of mg (Z)-9-tricosene standard, but smaller than that of 0.01 mg. Therefore, we diluted (Z)-9-tricosene in dichloromethane to concentrations closely similar to the component of the male body extract for titre analysis. We injected 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, and 0.01 mg of the synthetic sample to obtain a calibration regression equation. The quantity of the male-produced (Z)-9-tricosene from one male body extract was calculated by comparing the peak area with that of the synthetic standard sample. We calculated the quantity of the male-produced pheromone from one male body extract according to the calibration regression equation and the volume of the extract solution. (c) Electroantennogram (EAG) recording The olfactory chemoreceptors for airborne, volatile pheromones have not yet been determined in spiders (Foelix 1996). Odours of conspecific females and of prey species, however, evoke electrical reactions in male pedipalps (Gemeno et al. 2000; Tichy et al. 2001). Putative chemoreceptors on female spider legs have been examined by electron microscopy (Ross & Smith 1979; Barth 2002). Electroantennogram (EAG) responses to the synthetic tricosane and (Z)-9-tricosene were recorded from the first leg tarsus of sexually RFs in our experiment. One leg of the tested spider was excised at the proximal base of the tarsus. The tip of the terminal tarsus segment was removed. Both the proximal end and distal ends of the tarsus were stuck on the electrode holder (PRG22 probe; Syntech, Hilversum, The Netherlands) using electrically conductive gel. The electrode holder was connected to a high-impedance signal amplifier (Intelligent Data Acquisition Controller CS-55, Proc. R. Soc. B (2010)

9 Downloaded from rspb.royalsocietypublishing.org on August 26, 2010 Male-produced aphrodisiac in a spider Y.-H. Xiao et al choice chamber 1 release chamber choice chamber 2 Figure 1. Two-choice arena system used in the behavioural assays. The three chambers were upended to each other and spliced with a hole on one or two sides. The selecting spider was released in the central chamber and could move freely to the left or right choice chamber. Syntech). The amplified electrical potential signal from the antenna was recorded on a personal computer using the Syntech EAG software. An odour puff filtered through an active carbon filter was mixed with a humidified airstream blowing continuously over the tarsus preparation at a rate of 250 ml min 21 through a silicone rubber pipe connected to a glass tube terminating 3 mm from the tarsus preparation. Both tricosane and (Z)-9-tricosene were dissolved in dichloromethane at 0.1 mg ml 21. Ten microlitre aliquots of each tested compound or dichloromethane alone (control) were absorbed on pieces of filter paper (5 30 mm). The filter paper was inserted into the glass pipette and exposed to the air stream 8 min later, by which time the solvent had evaporated at room temperature (258C). The order of the odour stimuli was as follows: air (control), dichloromethane (solvent blank), tricosane, (Z)-9-tricosene, dichloromethane and air. The EAG responses were tested using five tarsus preparations each of a female P. beijingensis. All tests were repeated twice for each tarsus. (d) Behavioural assays First, we tested the attractiveness of (Z)-9-tricosene to sexually RFs in a two-choice arena system used in previous behavioural assays of male responses to the female-produced sex pheromone of P. beijingensis (Xiao et al. 2009). The three chambers were upended and spliced with a hole on one or two sides (figure 1). The test female was released in the central chamber and could move freely to the left or right choice chamber. Sexually RFs were separated into three groups to test the attractiveness of (Z)-9-tricosene, tricosane, and a blend of (Z)-9-tricosene and tricosene, respectively. (Z)-9- tricosene, tricosane and the binary blend were dissolved in dichloromethane at concentrations of 0.001, 0.01 and 0.1 mg ml 21. The test females were introduced into the central release chamber and allowed to acclimatize to the surroundings for 1 h before the trials. A piece of filter paper containing 10 ml of the test solution was placed in one of the choice chambers; the other choice chamber contained a filter paper with an equal quantity of dichloromethane (solvent control). The choice chambers contained the chemical solution or the control solvent alternately between successive trials to eliminate any possible bias. As searching behaviour of this spider only occurs in the dark (Y.-H. Xiao, J.-X. Zhang & S.-Q. Li 2008, unpublished data), these tests were completed at night (usually from to h). We observed movement of the test females under infrared light and recorded which chamber it chose first during a 2 h observation period. If any test female was disturbed and escaped into a choice chamber as soon as the choice arena system was connected, we discarded the trial. Each test female was used twice in the two-choice behavioural assay. We rested each female for one week before the second test trial. Second, we tested whether the sexually RFs could detect (Z)-9-tricosene as a cue of gender from the conspecific males and thus adjust their behaviour to mate with the males. In the laboratory experiments, males always held still on or under the female webs for a short time (usually from a few minutes to half an hour) after they had been introduced into the female webs. After a short interval for acclimation, the males started to move on the web to search for the female, which is the beginning of male courting sequences. As our former study mentioned (Xiao et al. 2009), male spiders of P. beijingensis display courting behaviour actively during most of the courtship sequence while females stay motionless on the web. When the male unfolds its pedipalps, the female turns from passive to active and approaches the male for mating. If females were not sexually receptive (e.g. subadult females or gravid adults), they would attack the intruders as soon as we introduced the male spiders on their webs. In a sense, keeping motionless (instead of showing aggressive behaviour) indicates the sexual acceptability of the females. Therefore, we compared male acclimation time, male courting time and mating time to illustrate differences between the treatment groups and the control group. Square boxes made of plastic-coated cardboard (22 22 cm; 8.5 cm deep) with transparent glass tops were used for observing spider courtship and copulation. All boxes were cleaned with 95 per cent ethanol and air dried before use. A glass dish with cotton soaked in distilled water was set in each box to supply humidity. Sexually RFs were randomly divided into four groups to observe the courting and mating behaviour of the males while exposed to (Z)-9-tricosene, tricosane, binary blends of (Z)-9-tricosene and tricosene, and dichloromethane (solvent control), respectively. Females were released into the box individually and were allowed to build their webs for 24 h prior to the trials. We placed a piece of filter paper with 10 ml of test solution at 0.1 mg ml 21 in each treated female box, while a filter paper with 10 ml dichloromethane alone was placed in each control female box. Twenty minutes later, MM spiders were introduced into all treated and control female boxes (one for each). For each trial, we recorded three parameters. (i) Male acclimating time, which is defined as the interval (min) between the male introduction into the female box and the male first moving on the web. During this period, the male holds still on or under the female web to acclimate to the surroundings. (ii) Male courting time, which is defined as the interval (min) between the male first moving on the web and mating initiation. (iii) Mating time, which is defined as the interval (min) between the male inserting his pedipalps into the female s genital pore until the mates disengaged. We observed 10 pairs in each treatment group and in the control group. Three pairs in the tricosane group and one pair in each other group failed to mate. Each female and male was used only once. Third, we tested whether the sexually RFs could detect (Z)-9-tricosene as a cue for the presence of prey, and adjust their behaviour to take aggressive action against the intruders efficiently. All test females were kept individually in a glass cuvette (4 cm i.d. 12 cm deep), in which they had lived for several days to acclimatize. No food was provided to the test spiders for 6 days before each trial. Proc. R. Soc. B (2010)

10 Downloaded from rspb.royalsocietypublishing.org on August 26, Y.-H. Xiao et al. Male-produced aphrodisiac in a spider The females were divided into two groups: a treatment group (n ¼ 17) and a control group (n ¼ 17). We placed a filter paper with 10 ml of(z)-9-tricosene at 0.1 mg ml 21 in each treated female cuvette, while a filter paper with 10 ml of dichloromethane solvent was placed in each control female cuvette. Twenty minutes later, 10 fruitflies were introduced into each treated and control female container. We observed predatory behaviour of the females for 2 h. The number of fruitflies caught by the females and the time that females spent on catching the first prey were recorded. Nine females in the test group and two females in the control group did not display predatory behaviour during the observation period. Each female was used only once. (e) Statistical analyses We measured the relative abundance of each compound by converting the peak area of a particular compound into a percentage of the summed peak areas from the 13 main GC peaks. First, we compared differences in relative abundance between the compounds extracted from MM and RF individuals; second, we compared quantitative differences between compounds from MM and SM spiders. These were analysed using either independent two-tailed Student s t-tests when the data were normally distributed or nonparametric Mann Whitney U-tests when the data were not normally distributed. Differences in EAG responses among various odour stimulus groups were analysed by one-way analysis of variance (ANOVA) with the least significant difference (LSD) test. In the first behavioural assay, we used the x 2 -test for goodness-of-fit to compare observed with expected counts for female choice data obtained from the two-choice experiment so that we could determine whether (Z)-9-tricosene or tricosane was attractive to the test females. In the second behavioural assay, the Kruskal Wallis test was used when comparing differences in male acclimation and courting time among the four groups: the (Z)-9-tricosene exposure female group, the tricosane-exposure female group, the binary blend exposure female group and the control group, which did not have normally distributed raw data. Mann Whitney U-tests were used for paired comparisons for courting time. Differences in the mating time among the four groups were analysed using one-way ANOVA as the raw data were normally distributed. In the third behavioural assay, differences in the time taken for catching the first prey between the (Z)-9-tricosene exposure female group and the solvent exposure female group were assessed using the Mann Whitney U-test. Differences in the numbers of prey eaten between the treatment group and the control group were analysed using independent two-tailed Student s t-tests. All statistical analyses were conducted using SPSS for Windows (v. 15.0; SPSS Inc., Chicago, IL, USA). 3. RESULTS (a) Chemical identification More than 20 different compounds in the whole-body extracts of P. beijingensis were detected by the GC MS. Most were straight-chain aliphatic compounds, including aldehydes, ketones, acids, alkenes and alkanes. We tentatively identified 13 compounds that eluted in less than 30 min by matching GC retention times and mass spectra with analogues in the mass spectral library (figure 2). GC detection showed that compound 9 was present in body extracts of MM spiders while absent from body extracts of RFs and SMs (table 1). Except for compounds that were qualitatively different between the body extracts of the three spider groups, we also performed quantitative analyses on relative abundances of the relevant compounds obtained from the body extracts (table 1). Compound 10 showed a significantly greater proportion in body extracts of MM spiders than that of RFs or SMs. Compounds 9 and 10 were tentatively identified as (Z)-9-tricosene and tricosane, respectively, according to their retention times and mass spectra and later confirmed with the synthetic sample after separation on HP5 MS and a DB wax column. The mass spectra of these natural products matched those of synthetic standards (figure 3). (b) EAG responses EAG responses of first leg tarsi of RF spiders to the putative pheromone components (Z)-9-tricosene and tricosane are shown in figure 4. The tarsus preparations showed strong responses to (Z)-9-tricosene. The mean EAG amplitude response to (Z)-9-tricosene (0.223 mv) was significantly higher (ANOVA with LSD test, p, 0.01) than responses to the air blank (0.022 mv), solvent control (0.028 mv) or tricosane (0.030 mv). Nevertheless, differences in EAG responses among the tricosane-stimulating group, the air-stimulation group and the chloromethane-stimulating group are not statistically significant (LSD test, p. 0.05). These results clearly demonstrate that (Z)-9-tricosene evoked significant EAG responses from the tarsi preparations of the sexually RFs, while tricosane seemed to be inactive. (c) Titre analyses We determined the quantity of (Z)-9-tricosene from the body extract of one male by comparing the peak GC MS area detected from the extract of an MM spider with that detected from the synthetic standard. The calibration regression equation is y ¼ 0.302x (r 2 ¼ 0.918, S ¼ 0.311, p, 0.001). We calculated the quantity of the male-produced pheromone from one male body extract according to the calibration regression equation and the volume of the extract solution. There were large individual variations in pheromone titre from MM spiders. The amount of (Z)-9-tricosene varied from to mg, with a mean value of mg (+ s.e.). (d) Behavioural responses of females In the two-choice arena trials, sexually RFs displayed no significant preference between the treated chamber and the solvent chamber in the trials of three chemical dosages: (Z)-9-tricosene: x 2 1 (0.01 mg) ¼ 0.474, p ¼ 0.491; x 2 1 (0.1 mg) ¼ 0.034, p ¼ 0.853; x2 1 (1 mg) ¼ 0.862, p ¼ 0.353; tricosane: x 2 1 (0.01 mg) ¼ 0.391, p ¼ 0.532; x 2 1 (0.1 mg) ¼ 0.143, p ¼ 0.705; x2 1 (1 mg) ¼ 0.034, p ¼ 0.853; and the binary blend: x 2 1 (0.01 mg) ¼ 0.615, p ¼ 0.433; x 2 1 (0.1 mg) ¼ 0.048, p ¼ 0.827; x 2 1 (1 mg) ¼ 0.727, p ¼ 0.394; figure 5). These negative results of the two-choice assay show that neither (Z)-9-tricosene nor tricosane is an attractant released by the male P. beijingensis for conspecific females. In the second behavioural assay, there was no significant difference in male acclimation time among the Proc. R. Soc. B (2010)

11 Downloaded from rspb.royalsocietypublishing.org on August 26, 2010 Male-produced aphrodisiac in a spider Y.-H. Xiao et al (a) abundance abundance (b) * (c) abundance retention time (min) Figure 2. Total ion chromatograms of the crude extract from (a) a mate-searching male, (b) a sexually receptive female and (c) a subadult male. A non-polar column HP5 MS (0.25 mm 30 m 0.25 mm) was used. The numbers that label the GC peaks correspond to peak numbers in table 1. The asterisk indicates peak 9 is specific in the chromatogram of mate-searching male extract. binary blend group, the (Z)-9-tricosene group, the tricosane group and the control group (x 2 1 ¼ 1.473, d.f. ¼ 3, p ¼ 0.689, Kruskal Wallis test; figure 6). In all successful mating pairs, average mating times of the mates in the three treatment groups were a little longer than those of the mates in the control group but there was no significant difference between them (F ¼ 1.632, d.f. 1 ¼ 3, d.f. 2 ¼ 30, p ¼ by one-way ANOVA; figure 6). Males in the binary blend group and the (Z)-9-tricosene group, however, spent much less time courting females than did males in the tricosane group and the control group (x 2 1 ¼ 8.407, d.f. ¼ 3, p ¼ 0.038, Kruskal Wallis test; figure 6).Thedifferenceofmalecourtingtimebetween the binary blend group and the (Z)-9-tricosene group was not significant (U ¼ , p ¼ 0.288, Mann Whitney U-test). This indicates that tricosane is not a vital component for stimulating sexual behaviour in females. In the third behavioural assay, females exposed to (Z)- 9-tricosene spent a mean of min in catching the first fruitfly, while females exposed to the solvent dichloromethane spent a mean of min. There was no significant difference between these times ( p ¼ by Mann Whitney U-test; table 2). This result indicates that females in the treatment group did not initiate predatory behaviour more quickly than did females in the control group. On the contrary, 88 per cent of females in the control group caught the fruitflies for food while just 47 per cent of females in the treatment group caught their prey during the 2 h observation period. Females in the control group caught a mean of 2.35 fruitflies for food during the 2 h period, while females in the test group caught a mean of only 1.0. This difference was statistically significant (t ¼ 2.380, d.f. ¼ 32, p ¼ 0.023, by Student s t-test; table 2). Proc. R. Soc. B (2010)

12 Downloaded from rspb.royalsocietypublishing.org on August 26, Y.-H. Xiao et al. Male-produced aphrodisiac in a spider (a) (b) m/z Figure 3. Mass spectra of (a) peak 9 and (b) peak 10 in the body extract of mate-searching males. They were identified as (Z)-9- tricosene and tricosane by comparing retention time and mass spectra with analogue in the mass spectra library (NIST 2002) and confirmed after separating the authentic standards on a non-polar column (HP5 MS) and a polar column (DB wax). Table 1. Comparison of relative abundance of compounds in body extracts of the spider P. beijingensis (mean + s.d.). (MM, mate-searching males; RF, sexually receptive females; SM, subadult males.) relative abundance (%) statistical significance ( p) peak no. retention time (min) compounds MM (n ¼ 8) RF (n ¼ 9) SM (n ¼ 9) MM versus RF MM versus SM tetradecanal heptadecene tetradecanoic acid a a hexadecanal heptadecanone a hexadecanoic acid tricosene a 8 b silaceous compound c,d (Z)-9-tricosene d,e tricosane b silaceous compound a a pentacosane f diisooctyl phthalate a p-values were tested by using the Mann Whitney U-test; others were tested by using the independent t-test. b The silaceous compound were not considered as compounds from the spider body extracts but contaminants from the GC column. c The compound (Z)-9-tricosene is specific in the body extract of mate-searching males. d The compound was verified with synthetic standard samples after separation with a non-polar column (HP52MS) and a polar column (DB2wax); other compounds were identified by comparison with spectra listed in the NIST Mass Spectral Library (Agilent Technologies 2002). e Relative abundance of the compound tricosane in the body extract of mate-searching males was significantly more than that in body extract of sexually receptive females or subadult males. f This compound is a plasticizer contaminant and probably came from the box that was used to contain the spider to build their web. 4. DISCUSSION Our study revealed that females of P. beijingensis exposed to (Z)-9-tricosene initiated mating with the males far more quickly than did females without exposure. MM individuals thus release (Z)-9-tricosene acting as a sexual stimulant for conspecific females. Males of many spider species have long been known to use chemical tactics that increase the probability of mating. For example, males release silk-bound semiochemicals for attracting females or inhibiting the courtship behaviours of conspecific males (Ross & Smith 1979; Yoshida & Suzuki 1981; Roland 1984; Rao Ayyagari & Tietjen 1987; Suter et al. 1987; Becker et al. 2005). (Z)-9-tricosene is, however, to our knowledge, the first identified male pheromone found in spiders. Based on the results of the behavioural assay, this pheromone acts like an aphrodisiac in that it increases the likelihood that a female will mate (figure 6). The MM individuals appear to release (Z)-9- tricosene to stimulate the sexual behaviour (copulation) of RF spiders so that they can mate more quickly (figure 6). It seems that, however, this aphrodisiac pheromone is not attractive to the females because the females showed no preference to the chambers containing (Z)-9- tricosene in the two-choice assay (figure 5). In nature, most male spiders leave their retreats or webs at maturity and start wandering around or spin their own nests right next to the potential mates (Foelix 1996). The MMs in many spider species are apparently attracted by female sex pheromones (Gaskett 2007). In contrast to males, female spiders usually apply the sit-and-wait tactic for mating. Female P. beijingensis remain motionless during most of the male courtship. Only after the males unfold Proc. R. Soc. B (2010)

13 Downloaded from rspb.royalsocietypublishing.org on August 26, 2010 Male-produced aphrodisiac in a spider Y.-H. Xiao et al EAG response (mv) a a a b** 0 air dichloromethane tricosane (Z)-9-tricosene Figure 4. EAG responses (mean + s.e.) of first leg tarsi (n ¼ 5) of sexually receptive female P. beijingensis to putative pheromone compounds. The order of the odour stimuli was as follows: air (control), dichloromethane (solvent blank), tricosane and (Z)-9-tricosene. **p, 0.01 (one-way ANOVA with LSD test). (a) 70 first female choice (%) (b) first female choice (%) μg μl 1 (n=21) μg μl 1 (n=32) μg μl 1 (n=31) (c) first female choice (%) μg μl 1 (n=28) μg μl 1 (n=30) μg μl 1 (n=30) μg μl 1 (n=30) μg μl 1 (n=26) μg μl 1 (n=25) Figure 5. Results of the attractiveness for female Pholcus beijingensis to (a)(z)-9-tricosene, (b) tricosane and the binary blends of (Z)-9-tricosene and (c) tricosane. Trials were completed in the two-choice arena system. (a) Black bars, (Z)-9-trocosene chamber; white bars, solvent chamber; striped bars, release chamber; (b) black bars, ticosane chamber; white bars, solvent chamber; striped bars, release chamber; (c) black bars, binary blends chamber; white bars, solvent chamber; striped bars, release chamber. Release chamber refers to females that within the 2 h observation period failed to leave the central arena into which they had been introduced 1 h prior to the start of the trial. their palps at the last phase of the courting procedure will the females turn from passive to active, which leads directly to copulation (Xiao et al. 2009). (Z)-9-tricosene has also been identified as a sex pheromone released by the female housefly Musca domestica (Carlson et al. 1971), which is a prey species of P. beijingensis. Later, some other compounds such as (Z)-9,10-epoxytricosane, (Z)-14-tricosene-10-one and some methyl alkanes were found to enhance the male housefly s sexual activity in combination with (Z)-9- tricosene (Uebel et al. 1976; Rogoff et al. 1980). A number of animal species use chemical camouflage to lure prey or access hosts (Dettner & Liepert 1994; Akino et al. 1999; Geiselhardt et al. 2006). A good example for chemical mimicry in the spiders is found among the bolas spiders. They usually feed exclusively Proc. R. Soc. B (2010)

14 Downloaded from rspb.royalsocietypublishing.org on August 26, Y.-H. Xiao et al. Male-produced aphrodisiac in a spider time (min) acclimating a* b b a* courting mating Figure 6. Influences of (Z)-9-tricosene, tricosane and the binary blend of both (Z)-9-tricosene and tricosane on courtship and mating behaviour of P. beijingensis. Black bars, binary blend; striped bars, (Z)-9-tricosene; dashed bars, tricosane; white bars, solvent control. Acclimating time is defined as the interval (min) between the male introduction into the female box and the male first moving on the web. In this period, the males keep still on the box side or bottom. Male courting time is defined as the interval (min) between the male first moving on the web and mating initiation. During this period, the male court the female until mating. Mating time is defined as the interval between the male inserting his pedipalps into the female s genital pore until the mates disengaged. Data are mean + s.e. *p, Table 2. Predatory behaviour of females exposed to (Z)-9-tricosene in comparison with that of females exposed to solvent only. Females exposed to (Z)-9-tricosene were in the test group and females exposed to solvent only (dichloromethane) were in the control group. test group predatory amount time on catching the first fruitflies control group predatory amount time on catching the first fruitflies x x 2.35 a a Significant difference between the average number of fruitflies caught by females in the control group and those caught by females in the test group. on males of a restricted number of moth species (Yeargan 1994). Adult female bolas spiders attract male moth prey by combinations of aggressive chemical mimicry with a specialized weapon (the bolas) and behaviour (Eberhard 1977; Stowe et al. 1987; Gemeno et al. 2000; Haynes et al. 2002). Cuticular hydrocarbons have also been reported as mimic chemicals in spiders. Thus, cuticular lipids of the spider Gamasomorpha maschwitzi, which lives in colonies of the Southeast Asian army ant Leptogenys distinguenda, and its host ant were virtually identical (Schulz 2004). This predatory spider acquires colony-specific cuticular hydrocarbons from their ant prey (Elgar & Allan 2004). The salticid spider Cosmophasis bitaeniata, which preys on the larvae of the green tree ant, Oecophylla smaragdina, mimics the cuticular hydrocarbon pattern of its host to avoid detection by major worker ants (Allan et al. 2002). In the study of Nentwig (1983), Pholcus showed high consumption rates of Coleoptera, Heteroptera, Hymenoptera Parasitica, Formicidae, Lepidoptera, Nuroptera, Orthoptera and Dermaptera. In our laboratory experiments, P. beijingensis accepted almost all offered prey species including fruitflies, springtails, mosquitoes, houseflies, ants and even conspecific spiderlings. In field investigations, several insect species and other small arthropods including mosquitoes (Chironomidae, Tipulidae, Cecidomyiidae), moths (Gelechiidae), ants (Formicidae), bedbugs (Coreidae), houseflies (Muscidae) and pillbugs (Porcellionidae) were captured by P. beijingensis (Y.-H. Xiao, J.-X. Zhang & S.-Q. Li 2009, unpublished data). Whether the male P. beijingensis mimics the sex pheromones of their prey needs further investigation and is beyond the scope of this paper. Hydrocarbons serve many functions in insects. They comprise a significant portion of the cuticular lipids that prevent desiccation, and are important in chemical communication (Howard & Blomquist 1982). (Z)-9-tricosene is a very common compound of cuticular hydrocarbons of insects in general. As well as in the housefly, (Z)-9- Proc. R. Soc. B (2010)

15 Downloaded from rspb.royalsocietypublishing.org on August 26, 2010 Male-produced aphrodisiac in a spider Y.-H. Xiao et al tricosene was also found in another fly species (Haematobia irritans) and males contained more than females (Macldey 1977). Intriguingly, (Z)-9-tricosene has been found in several other insects as a biologically active component. Zhang et al. (2003) identified five monounsaturated compounds including (Z)-9-tricosene from the Asian long-horned beetle, Anoplophora glabripennis and demonstrated that these compounds stimulate copulatory behaviour in males. In the social wasp, Vespa crabro, cuticular hydrocarbons including (Z)-9-tricosene are involved in the phenomenon of nestmate recognition (Ruther et al. 2002). Thom et al. (2007) reported that (Z)-9-tricosene along with three other hydrocarbons could be isolated from the scent of waggle-dancing foragers of the honeybee (Apis mellifera). These compounds are semiochemicals, inducing worker recruitment to the food source. It is not unique that the male spider of P. beijingensis shares its semiochemical with an insect species. Our previous study on the silk-bound pheromone of the spider P. beijingensis showed that the female sex pheromone components, (E,E)-farnesyl acetate and hexadecyl acetate, are found not only in some invertebrate species such as insects but also in mammals such as voles (Xiao et al. 2009). The spider pheromone 8-methyl-2-nonanone, isolated from the orb-web spider A. aperta (Papke et al. 2001), resembles a known pheromone component of the caddisfly, Hesperophylax occidentalis (Bjostad et al. 1996) and of the Asia palm weevil, Rhynchophorus ferrugineus (Hallett et al. 1993). 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17 Zoological Journal of the Linnean Society, 2010, 160, With 23 figures Intraspecific or interspecific variation: delimitation of species boundaries within the genus Gammarus (Crustacea, Amphipoda, Gammaridae), with description of four new species ZHONGE HOU and SHUQIANG LI* Institute of Zoology, Chinese Academy of Sciences, Beijing , China Received 19 November 2008; accepted for publication 11 May 2009 The delineation of Gammarus species is controversial because of extensive intraspecific morphological variation. The current study examined DNA sequences from the mitochondrial cytochrome c oxidase subunit I and the nuclear 28S genes as well as morphological and ecological data to determine the species boundaries of Gammarus species from China. The results of molecular analyses showed that Gammarus sp1, G. sp2, G. sp3, and G. sp4 are monophyletic and deeply divergent from sister groups. Detailed morphological and ecological comparisons with closely related species were consistent with molecular analyses. Gammarus sp1, G. sp2, G. sp3, and G. sp4 were described as four new species: Gammarus illustris sp. nov., Gammarus clarus sp. nov., Gammarus hypolithicus sp. nov., and Gammarus parvioculus sp. nov. We recommend that molecular detected species should be formally named and described for future research The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160, doi: /j x ADDITIONAL KEYWORDS: ecological data mitochondrial DNA nuclear DNA species concept taxonomy. INTRODUCTION Establishing species boundaries is fundamentally important in biodiversity assessment and in subsequent conservation strategy design. Nevertheless, at the practical level, the determination of species boundaries remains challenging to taxonomists. The traditional procedure of morphological delimitation is to compare a list of fixed diagnostic characters, which hypothetically reflects reproductive isolation amongst putative taxa (Wiens & Servedio, 2000); however, reliance on morphology alone cannot solve the problem of species with similar morphology (Meyran, Monnerot & Taberlet, 1997). Consequently, many systematists incorporate molecular data and use a phylogenetic approach to address the species boundary *Corresponding author. lisq@ioz.ac.cn problem. The phylogenetic species concept is applied in most biological studies that are concerned with evolutionary history (Cracraft, 1983). However, one major issue associated with the phylogenetic delimitation of species is how to determine the level of monophyly that is equivalent to species (Sanders, Malhotra & Thorpe, 2006). Monophyly exists at all levels within a phylogeny; selecting ancient or recent clades as species can give very different counts of species. A commonly used approach is to employ a genetic distance as a threshold to determine species status. For example, DNA barcoding employs ten times the mean intraspecific divergence to infer species diagnosis (Hebert et al., 2004; Witt, Threloff & Hebert, 2006). However, it remains doubtful as to whether universal divergence values exist amongst species (Zhang et al., 2008). Furthermore, molecular analyses have revealed numerous morphologically 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

18 216 Z. HOU and S. LI cryptic species (Witt et al., 2006). Consequently, molecular analyses have detected many cryptic species that are commonly designated as provisional species, such as SP1 and SP2, or lumped within a currently recognized species. A species with only molecular characters does not meet Article 16 in the International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature, 1999), and no new species has been described on molecular characters alone. Species designated as SP1 or SP2 cannot be accepted by biological projects (such as Species 2000 and Global Biodiversity Information Facility), which leads to bias in assessments of biodiversity (Guralnick, Hill & Lane, 2007). As morphological or molecular data separately do not necessarily address species boundaries correctly, combination of multiple types of independent sources of data, including molecular, morphological, and ecological data, is required to assess species boundaries (Sanders et al., 2006; Roe & Sperling, 2007). The genus Gammarus Fabricius, 1775 includes more than 200 species and is widely distributed throughout the Northern Hemisphere (Väinölä et al., 2008). The taxonomy of Gammarus remains unsatisfactory despite recent revisions of regional faunas (Karaman & Pinkster, 1977a, b, 1987; Hou, 2002) and many current taxonomic publications (Stock et al., 1998; Özbek, 2007). The morphology-based taxonomy of Gammarus requires investigation of numerous characters, and detailed morphological studies have shown that many variable traits are of limited utility because of phenotypic plasticity and ecological diversity (Pinkster, 1983). Molecular markers are common additional sources in such cases, as DNA data provide nearly unlimited numbers of informative characters and have different levels of variation. Since the 1990s, mitochondrial DNA sequences have been widely employed to investigate closely related species (Meyran, Gielly & Taberlet, 1998; Müller, 2000). Although mtdna sequence data can be very effective, mtdna is prone to incomplete lineage sorting, ancestral polymorphisms, and introgression as a result of hybridization, which can lead to incorrect species delimitation (Funk & Omland, 2003). As such, it is desirable to use mtdna markers in combination with independent nuclear loci to elucidate species boundaries (Kelly, MacIsaac & Heath, 2006; Witt et al., 2006). Hou, Fu & Li (2007) reconstructed the phylogeny of Gammarus and showed that Asian Gammarus was monophyletic as deduced from nuclear and mitochondrial markers. Several lineages were designated as provisional species, Gammarus sp1 (Gammarus illustris sp. nov.), G. sp2 (Gammarus clarus sp. nov.), G. sp3 (Gammarus hypolithicus sp. nov.), G. sp4 (Gammarus parvioculus sp. nov.), and G. sp5 (Gammarus preciosus Wang, Hou & Li, 2009), based solely on molecular data. These provisional species often occupy similar habitats to other gammarids and are morphologically indistinguishable from similar species. For example, G. illustris occurs near the type locality of Gammarus gonggaensis Hou, 2002, and these two gammarids are morphologically similar. Gammarus clarus, G. hypolithicus, G. parvioculus, and Gammarus nekkensis Uchida, 1935 are indistinct using morphological characters for pairwise comparisons, and they are all distributed around Beijing. Gammarus clarus was initially identified as the same morphospecies as G. nekkensis, and mixed populations of these two amphipods were encountered. Gammarus hypolithicus and G. parvioculus were found at the peak of Baishishan Mt. and in Tongdong Cave, respectively. Gammarus preciosus was considered as a new species by its flat uropod 3, but we are not sure whether this character falls within the morphological variation of Gammarus pengi Hou, 2002 or whether it is a fixed difference. After molecular analyses, G. preciosus was confirmed as a new species, and has recently been described based on exhaustive analyses of morphological data (Wang et al., 2009). In this study, we explore the utility of molecular data (including mtdna and nuclear sequences) to clarify Chinese Gammarus species boundaries, especially for the taxa for which morphological and taxonomical confusion has been most apparent. We first clustered populations into distinct groups based on molecular data, then tried to find diagnostic characters for each group. Distinct mitochondrial and nuclear clades that corresponded to morphologically identified groups or ecological taxa were tentatively designated as species. In addition, formal taxonomic descriptions and habitats are presented for four newly detected species. MATERIAL AND METHODS MATERIAL FOR MOLECULAR ANALYSIS Specimens of Gammarus were collected from 64 sites in China (Fig. 1, Table 1). Special emphasis was placed on dubious species (G. clarus, G. hypolithicus, G. parvioculus) and the widely distributed species G. nekkensis from a previous report (Hou et al., 2007). For G. hypolithicus and G. parvioculus, specimens were only collected in the type locality despite our careful searches around the type locality. Species of Sinogammarus Karaman & Ruffo, 1995 were included because we consider Sinogammarus to be part of Gammarus (Hou et al., 2007). Gammarus pulex (Linnaeus, 1758), endemic to Europe, and Gammarus tigrinus Sexton, 1939, distributed in Europe 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

19 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 217 Figure 1. Sample sites for the species of Gammarus from China. and North America, were selected as outgroups based on the phylogenetic analyses of Hou et al. (2007). DNA SEQUENCE ANALYSIS Genomic DNA was extracted from the heads of specimens using a standard phenol chloroform isoamyl (PCI) protocol (Hillis et al., 1996). A fragment of cytochrome c oxidase subunit I (COI) was amplified with universal primers LCO1490 (GGTCAACAAATCAT AAAGATATTGG) and HCO2918 (TAAACTTCAGGGT GACCAAAAAATCA) (Folmer et al., 1994). An approximately 1400-bp fragment of 28S rdna was amplified by the primers 28F (TTAGTAGGGGCGAC- CGAACAGGGAT) and 28R (GTCTTTCGCCCCTAT- GCCCAACTGA) (Hou et al., 2007). PCRs were performed in 25 ml volumes containing 0.15 ml Taq polymerase (5000 U/mL, Promega), 2.5 ml 10 PCR buffer (2.0 mm MgCl 2), 0.8 ml deoxyribonucleotide triphosphates (dntps, 10 mm), 1 ml of each primer (10 pmol/ml), 1 ml template DNA, and ml distilled water. PCR settings for amplifying COI sequences consisted of initial denaturing of 60 s at 94 C, five cycles of 30 s at 94 C, 90 s at 45 C, 60 s at 72 C, then 35 cycles of 30 s at 94 C, 90 s at 51 C, 60 s at 72 C, and a final 5 min extension at 72 C. PCR conditions for 28S fragments involved initial denaturing of 60 s at 94 C, followed by 35 cycles of 30sat94 C,45sat48 C,60sat72 C,anda5min extension at 72 C. PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) and were sequenced with ABI BigDye terminator sequencing protocols. Automated sequence outputs were imported into SEQUENCHER (version 4.5 demo edition, Gene Codes Corporation) for visual inspection of the chromatographs The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

20 218 Z. HOU and S. LI Table 1. Specimen information and GenBank accession numbers Species Locality Latitude/ longitude Voucher number GenBank accession numbers COI 28S Gammarus abstrusus Lushan, Sichuan, China N/ E IZCASIA0344 EF EF Gammarus accretus Chishui, Guizhou, China N/107 E IZCASIA0480 EF EF Gammarus brevipodus Bayanbulak, Xinjiang, China 43 N/84 6 E IZCASIA0437 EF Gammarus comosus Tongzhi, Guizhou, China N/ E IZCASIA0406 EF EF Gammarus Chishui, Guizhou, China N/ E IZCASIA0463 EF EF craspedotrichus Gammarus craspedotrichus Lushan, Jiangxi, China N/ E IZCASIA0682 EU708619* Gammarus curvativus Dechang, Sichuan, China N/ E IZCASIA0418 EF EF Gammarus curvativus Daocheng, Sichuan, China 29 N/ E IZCASIA0668 EU146925* EU708615* Gammarus decorosus Urumqi, Xinjiang, China N/87 36 E IZCASIA0079 EF EF Gammarus electrus Haidian, Beijing, China N/ E IZCASIA0302 EF EF Gammarus elevatus Jianchuan, Yunnan, China N/99 48 E IZCASIA0538 EF Gammarus emeiensis Lushan, Sichuan, China N/ E IZCASIA0343 EF EF Gammarus glabratus Dafang, Guizhou, China 27 N/ E IZCASIA0398 EF EF Gammarus gonggaensis Baoxing, Sichuan, China N/ E IZCASIA0028 EF EF Gammarus gonggaensis Baoxing, Sichuan, China N/ E IZCASIA0055 EF EF Gammarus gregoryi Yunlong, Yunnan, China N/99 18 E IZCASIA0579 EF EF Gammarus Danba, Sichuan, China N/ E IZCASIA0196 EF EF kangdingensis Gammarus koreanus Ji an, Jilin, China N/ E IZCASIA0336 EF EF Gammarus lacustris Fangshan, Beijing, China N/ E IZCASIA0323 EF EF Gammarus lacustris Hoh Xil, Qinghai, China N/93 48 E IZCASIA0340 EF EF Gammarus lacustris Hoh Xil, Qinghai, China N/93 48 E IZCASIA0339 EF Gammarus lacustris Fangshan, Beijing, China N/ E IZCASIA0326 EF Gammarus lacustris Yanqing, Beijing, China N/ E IZCASIA0292 EF Gammarus lacustris Altun, Xinjiang, China N/90 18 E IZCASIA0423 EF Gammarus lacustris Xainza, Tibet, China N/88 42 E IZCASIA0618 EF Gammarus lacustris Haixi, Qinghai, China N/97 12 E IZCASIA0624 EF Gammarus Lichuan, Hubei, China N/ E IZCASIA0335 EF EF lichuanensis Gammarus Lichuan, Hubei, China N/ E IZCASIA0604 EF EF lichuanensis Gammarus liui Shenmu, Shaanxi, China N/ E IZCASIA0548 EF Gammarus madidus Laiyuan, Hebei, China N/ E IZCASIA0310 EF Gammarus martensi Zhouzhi, Shaanxi, China N/ E IZCASIA0415 EF EF Gammarus montanus Zhaosu, Xinjiang, China 43 6 N/81 6 E IZCASIA0432 EF EF Gammarus nekkensis Mentougou, Beijing, China 40 N/ E IZCASIA0511 EF EF Gammarus nekkensis Mentougou, Beijing, China 40 N/ E IZCASIA0059 EF EF Gammarus nekkensis Mentougou, Beijing, China 40 N/ E IZCASIA0051 EF Gammarus nekkensis Yanqing, Beijing, China N/ E IZCASIA0314 EF Gammarus nekkensis Yangyuan, Hebei, China 40 6 N/114 6 E IZCASIA0355 EF Gammarus nekkensis Yangyuan, Hebei, China 40 6 N/114 6 E IZCASIA0683 EU146930* EU708621* Gammarus nekkensis Weixian, Hebei, China N/115 E IZCASIA EU708612* Gammarus nekkensis Weixian, Hebei, China N/115 E IZCASIA EU146922* EU708611* Gammarus pengi Tianjun, Qinghai, China N/99 E IZCASIA0509 EF Gammarus pexus Benxi, Liaoning, China N/ E IZCASIA0523 EF EF Gammarus qiani Zhaotong, Yunnan, China N/ E IZCASIA0413 EF EF Gammarus qiani Zhaotong, Yunnan, China N/ E IZCASIA0411 EU146923* EU708614* Gammarus riparius Xuanen, Hubei, China N/ E IZCASIA0510 EF EF Gammarus shanxiensis Yangcheng, Shanxi, China N/ E IZCASIA0519 EF The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

21 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 219 Table 1. Continued Species Locality Latitude/ longitude Voucher number GenBank accession numbers COI 28S Gammarus Shenmu, Shaanxi, China N/ E IZCASIA0512 EF shenmuensis Gammarus Jiuzhaigou, Sichuan, China N/ E IZCASIA0011 EF EF sichuanensis Gammarus sinuolatus Danba, Sichuan, China N/ E IZCASIA0195 EF EF Gammarus illustris Jiulong, Sichuan, China 29 6 N/ E IZCASIA0171 EF EF sp. nov. (sp1) Gammarus clarus Laiyuan, Hebei, China N/ E IZCASIA0315 EF EF sp. nov. (sp2) Gammarus Laiyuan, Hebei, China N/ E IZCASIA0317 EF EF hypolithicus sp. nov. (sp3) Gammarus Fangshan, Beijing, China N/ E IZCASIA0318 EF EF parvioculus sp. nov. (sp4) Gammarus Fangshan, Beijing, China N/ E IZCASIA EU708613* parvioculus sp. nov. (sp4) Gammarus preciosus Nanyang, Henan, China N/ E IZCASIA0570 EF (sp5) Gammarus preciosus Nanyang, Henan, China N/ E IZCASIA0571 EU708620* (sp5) Gammarus Benxi, Liaoning, China N/ E IZCASIA0524 EF EF stalagmiticus Gammarus takesensis Takes, Xinjiang, China N/81 E IZCASIA0425 EF EF Gammarus taliensis Dali, Yunnan, China N/ E IZCASIA0572 EF Gammarus tastiensis Yumin, Xinjiang, China N/82 54 E IZCASIA0563 EF EF Gammarus translucidus Suiyang, Guizhou, China N/ E IZCASIA0403 EF Sinogammarus chuanhui Meitan, Guizhou, China N/ E IZCASIA0666 EF EF Sinogammarus chuanhui Wuchuan, Guizhou, China N/ E IZCASIA0679 EU146931* EU708618* Sinogammarus chuanhui Nanchuan, Sichuan, China N/ E IZCASIA0018 EF EF Gammarus pulex Germany IZCASIA0675 EU146924* EU708616* Gammarus tigrinus the Netherlands N/ E IZCASIA0609 EF EF *GenBank accession numbers for this study. Accession numbers starting with EF were also generated in this study, but they were previously used in Hou et al. (2007). All sequences were aligned using ClustalX (Thompson et al., 1997) and adjusted by eye using MacClade 4.0 (Maddison & Maddison, 2000). Parsimony analyses of sequence data were implemented using PAUP 4.0b10 (Swofford, 2002). All characters were equally weighted and unordered. Alignment gaps were treated as missing data. All phylogenetically uninformative characters were excluded from analysis. Heuristic searches used 1000 random sequence addition replicates by tree bisection reconnection (TBR) branch swapping. Branch support was estimated with bootstrap analyses (Felsenstein, 1985) using 1000 replicates. Bayesian analyses were conducted using MrBayes (Ronquist & Huelsenbeck, 2003). The best-fit model was selected by hierarchical likelihood ratio tests using MODELTEST 3.7 (Posada & Crandall, 1998). Each Bayesian analysis was run for The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

22 220 Z. HOU and S. LI generations, sampling every 100 generations. After generations, the average split frequencies between the two searches started to approach zero, indicative of convergence. We therefore designated the first sample trees as burn-in and used the last trees for constructing the 50% majorityrule consensus tree and estimating the Bayesian posterior probabilities (BPP). MORPHOLOGICAL OBSERVATION Specimens were examined and dissected in 75% ethanol. Prior to dissection, the body length was recorded by holding the specimen straight and measuring the distance along the dorsal side of the body from the base of the first antennae to the base of the telson. All dissected appendages were mounted on slides according to the methods described by Holsinger (1967). Appendages were drawn using an Olympus BX51 compound microscope equipped with a drawing tube. All types and other material have been deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS), Beijing. RESULTS Fifty-two COI and 57 28S sequences were obtained for ingroup taxa and two outgroup taxa (Table 1). The 656-bp COI alignment contained 347 variable sites, of which 311 were parsimoniously informative. There was no indication that pseudogenes were amplified as no gaps or stop codons were found in amino acid translation with invertebrate mitochondrial code. The 1311-bp 28S alignment contained 423 variable sites, of which 246 were parsimoniously informative. The best-fit models estimated by MODELTEST were the general time reversible model with invariant sites and gamma distribution rate variation (GTR + I + G) for COI and Tamura-Nei with invariant sites and gamma distribution rate variation (TrN + I + G) and for 28S. Uncorrected pairwise divergences between ingroup taxa ranged between 1.96 and 32.7% for COI and between 0.1 and 11.3% for the 28S gene. Maximum parsimony (MP) analysis of COI revealed 100 most parsimonious trees with a length of 2603, a consistency index (CI) of , and a retention index (RI) of MP analysis of 28S revealed 4798 most parsimonious trees with a length of 1219, a CI of , and a RI of Bayesian analysis produced trees for the COI (Fig. 2) and 28S (Fig. 3) datasets, with congruent topologies with MP strict consensus trees. Specimens that were morphologically identified as G. nekkensis were clustered into a distinct clade. Both COI and 28S Bayesian analyses supported G. nekkensis from voucher numbers 337-1, 337-2, 51, 59, and 511 forming a distinct cluster, which agreed with the descriptions and illustrations of G. nekkensis (Karaman, 1989). These were designated as true G. nekkensis. Other specimens of G. nekkensis (voucher numbers 314, 355, 683) were supported as a monophyletic group with G. clarus. The uncorrected pairwise divergence between G. clarus and the true G. nekkensis was % for COI and % for 28S. The differentiation amongst G. hypolithicus, G. parvioculus, and G. nekkensis was more than 15% for COI and 2 4% for 28S. Specimen 171 (G. illustris) was initially identified as G. gonggaensis based on morphology; however, on the molecular tree, it was the sister group of the clade Gammarus curvativus Hou & Li, 2003 and Gammarus gregoryi Tattersall, 1924, with 28S divergence of 9 and 9.79%. The divergence between G. illustris and other ingroup species ranged from 6.71 to 10.3%, averaged at 8.11% for 28S. DISCUSSION Molecular analyses showed that G. illustris was grouped with the clade of G. gregoryi and G. curvativus (Fig. 3). However, the divergences of 9% for the nuclear gene 28S between G. illustris and the clade of G. gregoryi and G. curvativus exceeded the maximum distance (5%) for another amphipod taxa (Hyalella) (Witt et al., 2006), which suggests a long period of evolutionary independence. On morphology, G. illustris differs from G. gregoryi in pereopod 3 with long setae on the posterior margin and urosomite 1 with setae on the dorsal margin but no spines. In addition, these species have allopatric distributions: G. illustris is located at high altitudes, above 3500 m, whereas G. gregoryi and G. curvativus are distributed below 2000 m altitude. Therefore, molecular, morphological, and ecological data support G. illustris being a new species. The results of this study reveal significant mitochondrial differentiation (> 15%) for the comparisons amongst G. clarus, G. hypolithicus, G. parvioculus, and G. nekkensis. Various COI distances used for cryptic species have been previously reported for Gammarus species, such as 4% for Gammarus duebeni (Rock et al., 2007) and 8 11% for G. tigrinus (Kelly et al., 2006). The value of 15% divergence for G. clarus, G. hypolithicus, G. parvioculus, and G. nekkensis is sufficient to recognize these taxa as new species. Gammarus clarus was supported as monophyletic and divergent from G. nekkensis at both mitochondrial and nuclear loci. At the same time, we found that combinations of several morphological traits, such as elevation of urosomites and both rami of uropod 3 with plumose setae, distinguish G. clarus from G. nekkensis. It seems appropriate to maintain 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

23 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 221 Figure 2. The 50% majority rule consensus tree from the Bayesian analysis of the Gammarus cytochrome c oxidase subunit I data set. Numbers above the lines are Bayesian posterior probabilities. Sinogammarus species are underlined. Voucher numbers follow the species names The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

24 222 Z. HOU and S. LI Figure 3. The 50% majority rule consensus tree from the Bayesian analysis of the Gammarus 28S data set. Numbers above the lines are Bayesian posterior probabilities. Sinogammarus species are underlined. Voucher numbers follow the species names The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

25 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 223 G. clarus as a species. However, we found that distributions of G. clarus and G. nekkensis overlap, so isolation by distance is not the only barrier restricting gene flow using vicariance terminology. Other factors such as mating behaviour, pheromone preference, water PH value, and other ecological factors could all contribute to this speciation (Roe & Sperling, 2007), although these need to be further explored. Molecular analyses indicated that G. hypolithicus was genetically divergent from G. clarus and G. nekkensis, and could be morphologically distinguished by a combination of characters, such as the presence of setae of pereopod 3 and uropod 3. Moreover, we found no evidence of ecological compatibility for G. hypolithicus with other Gammarus species, as G. hypolithicus was only found in the type locality at a high altitude despite our intensive survey. Gammarus hypolithicus may be confined to a narrow ecological range because of competition with the more widely distributed Gammarus species G. clarus and G. nekkensis at low altitude. The molecular data show that G. parvioculus is nested in a clade containing G. nekkensis, G. clarus, and G. hypolithicus, and is genetically distinct from the remaining species (Figs 2, 3). Gammarus parvioculus is restricted to caves, and possesses troglobitic characters: reduction of eyes and slender appendages. Therefore, the combination of molecular, morphological, and ecological data support the assumption that G. parvioculus evolved independently, and geographical barriers restricted gene flow between the cave and surface gammarids. Delimiting species boundaries is not straightforward because of disagreements about the theoretical concept of a species based on different aspects of organismal biology (de Queiroz, 2007). This problem has been demonstrated in Gammarus species (Meyran et al., 1997). In the current study, G. clarus, G. hypolithicus, G. parvioculus, and G. nekkensis show little morphological variation, whereas the genetic divergence is high (Figs 2, 3). These gammarids proved monophyletic in molecular analyses, which indicated that a diverse combination of morphological diagnostic characters is required to distinguish amongst them. In addition, G. hypolithicus and G. parvioculus inhabit separate habitats. Molecular analyses integrated with morphological and ecological data confirmed that they are new species. In recent years, increasing numbers of morphologically cryptic species have been detected in molecular and ecological analyses, a development that started in the 1980s (Scheepmaker, 1987). Rock et al. (2007) reported that based on mitochondrial information G. duebeni Liljeborg, 1852 diverged into two lineages, with a freshwater and a marine form. A phylogeographical study of the estuarine G. tigrinus Sexton, 1939, widespread in North America, supported the Pleistocene separation into northern and southern clades (Kelly et al., 2006). However, few of these cryptic Gammarus species are formally described and named, a result of the lack of discriminating morphological features. Molecular and ecological analyses help us to resolve the species delimitation of Gammarus species despite the overlap of intra- and interspecific morphological character variations. Other amphipod species boundaries will be determined with the use of molecular analyses and ecological data, especially in subterranean or cave species, most of which are virtually indistinguishable morphologically (Lefébure et al., 2006). No doubt molecular species delimitation, in combination with morphological analyses, will improve the effectiveness of biodiversity assessment in relation to ecological and conservation biological issues. SYSTEMATICS GENUS GAMMARUS FABRICIUS, 1775 Diagnosis: Antenna 1 longer than antenna 2, primary flagellum longer than peduncle, accessory flagellum with more than two articles. Maxilla 1 with 11 apical spines on outer plate, palps asymmetric. Maxilla 2 with a diagonal row of plumose setae on inner plate. Gnothpods with one or more midspines on the palmar margin of propodus. Uropod 3 with two articles for outer ramus. Telson deeply cleft. Coxal gills present on gnathopod 2 and pereopods 3 7. Remarks: Gammarus as one of the largest genera of Amphipoda, is widely distributed throughout the Northern Hemisphere. Most of the species are epigean freshwater taxa, with a few species reported from subterranean habitats and coastal marine waters. Barnard & Barnard (1983) reviewed the freshwater Amphipoda worldwide and 117 species were listed under the genus Gammarus. Since then, some regional works of the taxonomy of Gammarus have been undertaken, such as Gammarus species from Iran (Khalaji-Pirbalouty & Sari, 2004) and a Gammarus list from Turkey (Özbek & Balik, 2009). Up to now, more than 200 Gammarus species have been recorded (Väinölä et al., 2008). GAMMARUS ILLUSTRIS SP. NOV. (FIGS 4 8) Synonymy: Gammarus sp1 Hou et al., 2007: 599. Material examined: Holotype (IZCAS-I-A171), male, Wuxu Lake (29 6 N, E), altitude 3600 m, Jiulong County, Sichuan Province, collected by Z. Hou and Y. Lin, 26.vii Paratypes (from IZCAS-I- A171-2 to IZCAS-I-A171-26): 12 males, nine females, and four juveniles, same locality as holotype The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

26 224 Z. HOU and S. LI Figure 4. Gammarus illustris sp. nov., holotype, male. A, head; B, antenna 1; C, antenna 2; D, upper lip; E, left mandible; F, incisor of right mandible; G, lower lip; H, left maxilla 1; I, outer plate of left maxilla; J, palp of right maxilla; K, maxilla 2; L, maxilliped The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

27 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 225 Figure 5. Gammarus illustris sp. nov., holotype, male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2; E, epimeral plate 1; F, epimeral plate 2; G, epimeral plate 3; H, pleopod 1; I, pleopod 2; J, pleopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

28 226 Z. HOU and S. LI Figure 6. Gammarus illustris sp. nov., holotype, male. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, dactylus of pereopod 3; G, dactylus of pereopod 4; H, dactylus of pereopod 5; I, dactylus of pereopod 6; J, dactylus of pereopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

29 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 227 Figure 7. Gammarus illustris sp. nov., male, A E; female, F K. A, urosomites 1 3 (dorsal view); B, uropod 1; C, uropod 2; D, uropod 3; E, telson; F, pereopod 3; G, oostegite of gnathopod 2; H, oostegite of pereopod 3; I, oostegite of pereopod 4; J, oostegite of pereopod 5; K, telson The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

30 228 Z. HOU and S. LI Figure 8. Gammarus illustris sp. nov., female. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2; E, uropod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

31 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 229 Etymology: The specific name illustris is derived from the name of the type locality, Wuxu Lake, meaning shining lake in Tibetan. Diagnosis: Calceoli absent on antenna 2; posterodistal corner of epimeral plates 1 3 with small acute tooth; urosomite 1 with setae only on dorsal margin and no spines; inner ramus of uropod 3 reaching 0.2 times the length of outer ramus, both inner and outer ramus densely set with simple setae. Description Holotype: male Body length: 8.5 mm. Head (Fig. 4A): eyes oval, 1.6 times as long as wide; inferior antennal sinus distinct, ventral recessed margin nearly straight. Antenna 1 (Fig. 4B): peduncular articles 1 3 in length ratio 1 : 0.8 : 0.5, with lateral and distal setae; flagellum with 22 articles, aesthetascs present from articles 5 20; accessory flagellum with three articles; both primary and accessory flagella with distal setae. Antenna 2 (Fig. 4C): peduncular article 4 about as long as article 5, both with three to four groups of long setae along anterior and posterior margins; flagellum with 11 articles, calceoli absent. Upper lip (Fig. 4D): convex, with minute setae. Left mandible (Fig. 4E): incisor with five teeth; lacinia mobilis with four teeth; spine row with five pairs of setae; molar with one plumose seta; palp article 2 with 12 marginal setae, article 3 about 0.85 times as long as article 2, with three A-setae on outer surface, three B-setae on inner surface, a row of 16 D-setae on posterior margin, and four E-setae apically. Right mandible (Fig. 4F): incisor with four teeth, lacinia mobilis bifurcate, with small teeth. Lower lip (Fig. 4G): inner lobe absent. Maxilla 1 (Fig. 4H J): asymmetrical, left inner plate with a row of 11 plumose setae; outer plate with 11 serrated apical spines, each spine with small teeth; second article of palp with five slender spines and three stiff setae on medial surface; right palp wider, article 2 with four stout spines, one pectinate spine, and one seta. Maxilla 2 (Fig. 4K): inner plate with a row of ten plumose setae and with medial setal row; outer plate with apical setae and setules on lateral margin. Maxilliped (Fig. 4L): inner plate with three stout apical and one subapical spines; outer plate with nine blade spines, six pectinate spines; palp article 4 hooked, with a group of setae at hinge of unguis. Gnathopod 1 (Fig. 5A, C): coxal plate subrectangular, with three setae on anterior corner; basis with long setae on anterior and posterior margins; carpus short, 1.5 times as long as wide, about 0.8 times as long as propodus; propodus oval, palm oblique, with one medial spine and nine spines on posterior margin; dactylus with one seta on outer margin and two short setae at hinge of unguis. Gnathopod 2 (Fig. 5B, D): coxal plate with three setae on anterior corner and one seta on posterior corner; basis with long setae along anteroproximal and posterior margins; carpus with parallel margins, 1.9 times as long as wide, about 0.7 times as long as propodus; propodus subrectangular, palm margin subacute, with one medial spine and two spines on medial posterodistal corner and two spines on lateral posterodistal corner; dactylus with one seta on outer margin and one seta near the hinge of unguis. Pereopod 3 (Fig. 6A, F): coxal plate with three setae on anterior corner and one seta on posterior corner; basis with long setae on posterior margin; merus and carpus densely set with long and weakly curled setae on posterior margin, merus with two spines accompanied by setae on anterior margin, carpus and propodus with spines on posterior margin; dactylus with one plumose seta on posterior margin, and two setae at hinge of unguis. Pereopod 4 (Fig. 6B, G): coxal plate with three setae on anterior corner, and four setae on posterior margin, posterior margin excavated; basis with groups of long setae on posterior margin; merus with three groups of straight setae on posterior margin, carpus and propodus with spines on posterior margin; dactylus with one plumose seta on posterior margin and two stiff setae at hinge of unguis. Pereopods 5 7 slender. Pereopod 5 (Fig. 6C, H): coxal plate with one seta on anterior corner and four setae on posterodistal corner; basis posterior margin straight, posterior corner subquadrate, with two long setae and six spines on anterior margin, a row of 11 setae on posterior margin; merus with two groups of setae on anterior margin, and two spines on posterior margin; carpus with two groups of spines on anterior margin and two groups of spines on posterior margin; propodus with three groups of spines on anterior margin and two groups of setae or spine on posterior margin; dactylus with one seta on posterior margin and two setae at hinge of unguis. Pereopod 6 (Fig. 6D, I): coxal plate with three setae on posterodistal corner; basis elongate, with two groups of long setae and five spines on anterior margin, posterior margin subsigmoidal, with a row of ten setae; merus to propodus with groups of spines accompanied by short setae on anterior margin; dactylus with one plumose seta on posterior margin and two setae at hinge of unguis. Pereopod 7 (Fig. 6E, J): coxal plate with four setae on posterior margin; basis with long setae and a row of five spines on anterior margin, posterior margin expanded, shape convex, with 12 setae on posterior 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

32 230 Z. HOU and S. LI margin, inner surface with two setae; merus to propodus with two to four groups of spines accompanied by few short setae on anterior margin, propodus with two setae on posterior margin; dactylus with one plumose seta on posterior margin and two stiff setae at hinge of unguis. Coxal gills: coxal gill of gnathopod 2 and gills of pereopods 3 5 a little shorter than bases; gill of pereopod 6 about half length of basis; gill of pereopod 7 smallest, less than half of basis. Epimeral plates (Fig. 5E G): epimeron 1 ventrally rounded, with three long and two stiff setae on anterior corner, and two setae on posterior margin, posterodistal corner with small acute tooth; epimeron 2 with one long seta on anterior corner, one spine accompanied by one seta on ventral margin, posterodistal corner with small acute tooth, with four setae on posterior margin; epimeron 3 with one long seta and two spines on anterior corner, posterodistal corner with small acute tooth, with three setae on posterior margin. Pleopods 1 3 subequal (Fig. 5H J), peduncle with long facial and marginal setae, with two retinacula accompanied by one to two setae on anterodistal corner; both inner and outer rami fringed with plumose setae. Urosomites 1 3 dorsally flat (Fig. 7A), urosomite 1 with four groups of long setae on dorsal margin, without spines; urosomite 2 with four spines accompanied by long setae on dorsal margin; urosomite 3 with one spine accompanied by long setae on each side and two setae on dorsal margin. Uropod 1 (Fig. 7B): peduncle with one basofacial spine, one spine on outer margin, two spines on inner margin, with one and two spines on inner and outer distal corners, respectively; outer ramus with one spine on outer margin and one spine on inner margin; inner ramus with one spine on inner margin, both rami with five distal spines. Uropod 2 (Fig. 7C): peduncle with one spine on inner and outer corners, respectively; outer ramus with one spine on outer margin; inner ramus with one spine on inner margin. Uropod 3 (Fig. 7D): peduncle with four short setae on lateral margin; inner ramus 0.6 times as long as peduncle, reaching 0.2 times the length of outer ramus, with one spine on lateral margin and one distal spine; article 1 of outer ramus with one single spine and two pairs of spines on outer margin, and two pairs of spines on distal margin, both margins densely set with simple setae, terminal article longer than adjacent spines. Telson (Fig. 7E): deeply cleft, 0.9 times as long as wide, each lobe with one distal spine accompanied by long setae and long setae on lateral surface. Female: paratype (IZCAS-I-A171-2) Body length: 8.1 mm. Gnathopod 1 (Fig. 8A, C): coxal plate with three setae on anterior corner and one seta on posterior margin; propodus oval, palm margin not as oblique as that of male, with six spines on posterodistal corner; dactylus with one seta on outer margin and three setae at hinge of unguis. Gnathopod 2 (Fig. 8B, D): propodus subrectangular, palm margin subacute, with three spines on posterodistal corner; dactylus with one seta on outer margin and two setae at hinge of unguis. Pereopods 3 and 4 with straight setae on posterior margin (Fig. 7F). Uropod 3 (Fig. 8E): inner ramus about 0.27 times as long as outer ramus, both rami with simple setae, terminal article of outer ramus longer than adjacent spines. Telson (Fig. 7K): cleft, 0.7 times as long as wide, each lobe with two distal spines accompanied by long setae, a pair of short and a pair of long setae on the surface. Oostegites (Fig. 7G J): oostegite of gnathopod 2 broad, with marginal setae, oostegite of pereopods 3 and 4 elongate, oostegite of pereopod 5 smallest. Habitat: The species occurs under detritus, where water flows through from peak of mountain. Remarks: Gammarus illustris sp. nov. can be distinguished from G. curvativus by the following characters (G. curvativus in parentheses): calceoli absent on antenna 2 (present); propodus of gnathopod 2 with long straight setae on dorsal margin (curled setae); urosomite 1 with setae on dorsal margin but no spines (with spines). This new species differs from G. gregoryi (state in parentheses) in calceoli absent (present); pereopod 3 with long setae on posterior margin (few setae on pereopod 3); urosomite 1 with four groups of setae (two groups of setae). GAMMARUS CLARUS SP. NOV. (FIGS 9 13) Synonymy: Gammarus sp2 Hou et al., 2007: 599. Material examined: Holotype (IZCAS-I-A315), male, headwaters of Jumahe River (39 18 N, E), altitude 836 m, Laiyuan County, Hebei Province, collected by Z. Hou and Y. Lin, 5.ix Paratypes (from IZCAS-I-A315-2 to IZCAS-I-A315-14): eight males and five females, same locality as holotype. Other material: 125 males and 108 females (from IZCAS-I-A314-1 to IZCAS-I-A ) (voucher number 314), Baihe River (40 36 N, E), lower river of Baidaohe Reservoir, Yanqing County, Beijing, 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

33 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 231 Figure 9. Gammarus clarus sp. nov., male. A, head; B, antenna 1; C, antenna 2; D, upper lip; E, left mandible; F, incisor of right mandible; G, lower lip; H, left maxilla 1; I, palp of right maxilla 1; J, maxilla 2; K, maxilliped The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

34 232 Z. HOU and S. LI Figure 10. Gammarus clarus sp. nov., male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2; E, epimeral plate 1; F, epimeral plate 2; G, epimeral plate 3; H, urosomites 1 3 (dorsal view); I, urosomites 1 3 (lateral view); J, telson The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

35 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 233 Figure 11. Gammarus clarus sp. nov., male. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, dactyle of pereopod 7; G, dactyle of pereopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

36 234 Z. HOU and S. LI Figure 12. Gammarus clarus sp. nov., male, A F; female, G J. A, pleopod 1; B, pleopod 2; C, pleopod 3; D, uropod 1; E, uropod 2; F, uropod 3; G, oostegite of gnathopod 2; H, oostegite of pereopod 3; I, oostegite of pereopod 4; J, oostegite of pereopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

37 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 235 Figure 13. Gammarus clarus sp. nov., female. A, antenna 2; B, gnathopod 1; C, gnathopod 2; D, propodus of gnathopod 1; E, propodus of gnathopod 2; F, telson The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

38 236 Z. HOU and S. LI collected by S. Li and Z. Hou, 7.iv Fifteen males and 12 females (from IZCAS-I-A355-1 to IZCAS-I- A355-27) (voucher number 355), a brook from Hutouliang Village (40 6 N, E), Yangyuan County, Hebei Province, collected by S. Li, 12.v Etymology: The specific name clarus is Latin for clear, referring to the habitat with clear water and no signs of pollution. Diagnosis: Peduncular articles of antenna 2 with short setae, calceoli present; pereopods 3 and 4 with long straight setae on posterior margins; basis of pereopods 6 and 7 proximally broad, distally narrow; epimeral plates 2 3 acute in posterodistal corners; urosomites 1 2 weakly elevated; inner ramus of uropod 3 about 0.7 times the length of the outer ramus, both rami of uropod 3 densely set with plumose setae. Description Holotype: male Body length: 13.5 mm. Head (Fig. 9A): eyes reniform, 2.2 times as long as wide; inferior antennal sinus distinct, ventral recessed margin curving. Antenna 1 (Fig. 9B): peduncular articles 1 3 in length ratio 1 : 0.8 : 0.4, with short lateral and distal setae; flagellum with 28 articles, aesthetascs present except for the last two articles; accessory flagellum with four articles; both primary and accessory flagella with short distal setae. Antenna 2 (Fig. 9C): peduncular article 4 about as long as article 5, both with three to four groups of setae along anterior and posterior margins, setae shorter than the width of peduncles; flagellum with 13 articles, calceoli present. Upper lip (Fig. 9D): convex, with minute setae. Left mandible (Fig. 9E): incisor with five teeth; lacinia mobilis with four teeth; spine row with seven pairs of setae; molar with one plumose seta; palp article 2 with 12 marginal setae, article 3 about 0.73 times as long as article 2, with five A-setae, two groups of B-setae (with two and four setae, respectively), a row of 21 D-setae, and five E-setae. Right mandible (Fig. 9F): incisor with four teeth, lacinia mobilis bifurcate, with small teeth. Lower lip (Fig. 9G): inner lobe absent. Maxilla 1 (Fig. 9H, I): asymmetrical, left inner plate with a row of 16 plumose setae; outer plate with 11 serrated apical spines, each spine with small teeth; second article of palp with seven slender spines and three stiff setae on medial surface; right palp wider, article 2 with five stout spines, one pectinate spine, and one seta. Maxilla 2 (Fig. 9J): inner plate with a row of 17 plumose setae and many setules on medial surface; outer plate with stiff apical setae and setules on lateral margin. Maxilliped (Fig. 9K): inner plate with three stout apical and one subapical spines; outer plate with 13 blade spines and three pectinate spines; palp article 4 hooked, with a group of setae at hinge of unguis. Gnathopod 1 (Fig. 10A, C): coxal plate subrectangular, with two setae on anterior corner and one seta on posterior corner; basis with long setae on anterior and posterior margins; carpus short, 1.4 times as long as wide, about 0.7 times as long as propodus; propodus oval, palm oblique, with one medial spine and 21 spines on posterior margin; dactylus with one seta on outer margin. Gnathopod 2 (Fig. 10B, D): coxal plate with three setae on anterior corner and one seta on posterior corner; basis with long setae along anteroproximal and posterior margins; carpus with parallel margins, 1.4 times as long as wide, 0.7 times as long as propodus; propodus subrectangular, palm margin subacute, with one medial spine and three spines on medial posterodistal corner and three spines on lateral posterodistal corner; dactylus with one seta on outer margin. Pereopod 3 (Fig. 11A): coxal plate with three setae on anterior corner and one seta on posterior corner; basis with long setae on posterior margin; merus densely set with long and straight setae on posterior margin, merus with spines accompanied by setae on anterior margin, carpus and propodus with spines and long straight setae on posterior margin; dactylus with one plumose seta on posterior margin and two setae at hinge of unguis. Pereopod 4 (Fig. 11B, G): coxal plate with three setae on anterior corner and four setae on posterior margin, posterior margin excavated; basis with groups of long setae on posterior margin; merus to propodus with straight setae on posterior margin, carpus and propodus with spines on posterior margin; dactylus with one plumose seta on posterior margin and two stiff setae at hinge of unguis. Pereopods 5 7 slender. Pereopod 5 (Fig. 11C): coxal plate with one seta on anterior corner and three setae on posterodistal corner; basis posterior margin straight, posterior corner subquadrate, with long setae and four spines on anterior margin, a row of ten setae on posterior margin; merus to propodus with spines along anterior and posterior margins accompanied by few short setae; dactylus with one seta on posterior margin and one seta at hinge of unguis. Pereopod 6 (Fig. 11D): coxal plate with one seta on anterior corner and one seta on posterodistal corner; basis elongate, with one group of long setae and three 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

39 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 237 spines on anterior margin, posterior margin dwindled distally and projected on posterodistal corner, with a row of nine setae; merus to propodus with groups of spines accompanied by short setae along anterior and posterior margins; dactylus with one plumose seta on posterior margin and one seta at hinge of unguis. Pereopod 7 (Fig. 11E, F): coxal plate with two setae on anterior margin and four setae on posterior margin; basis with a group of five long setae and a row of four spines on anterior margin, posterior margin weakly expanded with nine setae on posterior margin, posterodistal corner subquadrate, inner surface with one spine accompanied by three setae; merus to propodus with two to four groups of spines accompanied by few short setae on anterior margin, propodus with two groups of setae and one spine on posterior margin; dactylus with one plumose seta on posterior margin and one stiff seta at hinge of unguis. Coxal gills: coxal gill of gnathopod 2 and gills of pereopods 3 5 a little shorter than bases; gill of pereopod 6 about half length of basis; gill of pereopod 7 smallest, less than half of basis. Epimeral plates (Fig. 10E G): epimeron 1 ventrally rounded, with six long setae and two spines on anterior corner and two setae on posterior margin; epimeron 2 with two subventral spines and three spines on ventral margin, posterodistal corner weakly acute, with three setae on posterior margin; epimeron 3 with three long setae on anterior margin, four spines on ventral margin, posterodistal corner acute, with three setae on posterior margin. Pleopods 1 3 subequal (Fig. 12A C), peduncle with long facial and marginal setae, with two retinacula accompanied by one to two setae on anterodistal corner; both inner and outer rami with articles, fringed with plumose setae. Urosomites 1 and 2 weakly elevated (Fig. 10H, I), urosomite 1 with spines accompanied by short setae on dorsal margin; urosomite 2 with spines accompanied by short setae; urosomite 3 with spines accompanied by setae. Uropod 1 (Fig. 12D): peduncle with one basofacial spine, three spines on outer margin, three spines on inner margin, and two and one spines on outer and inner distal corners, respectively; outer ramus with one spine on outer margin and two spines on inner margin; inner ramus with two spines on inner margin, both rami with five distal spines. Uropod 2 (Fig. 12E): peduncle with two and three spines on outer and inner margins, respectively, one spine on inner and outer corners, respectively; outer ramus with two spines on outer and inner margins, respectively; inner ramus with two and one spines on outer and inner margins, respectively. Uropod 3 (Fig. 12F): peduncle with lateral setae and distal spines; inner ramus 1.8 times as long as peduncle, reaching 0.7 times the length of outer ramus, with two spines on lateral margin and three distal spines; article 1 of outer ramus with one single spine and three pairs of spines on outer margin, and two pairs of spines on distal margin, both margins densely set with plumose and simple setae, terminal article longer than adjacent spines. Telson (Fig. 10J): deeply cleft, 1.2 times as long as wide, each lobe with two distal spines accompanied by long setae, one basolateral spine and several short setae on lateral surface. Female, paratype (IZCAS-I-A315-2) Body length 13.2 mm, with 36 eggs. Antenna 2 (Fig. 13A): peduncular articles 4 and 5 with setae along inner margin, setae longer than the width of peduncle. Gnathopod 1 (Fig. 13B, D): coxal plate with three setae on anterior corner and one seta on posterior margin; propodus oval, palm margin not as oblique as that of male, with 14 spines on posterior margin; dactylus with one seta on outer margin. Gnathopod 2 (Fig. 13C, E): propodus subrectangular, palm margin subacute, with four spines on posterodistal corner; dactylus with one seta on outer margin. Pereopods 3 and 4 with straight setae on posterior margin. Uropod 3: inner ramus about 0.7 times as long as outer ramus, both rami with plumose setae, terminal article of outer ramus longer than adjacent spines. Telson (Fig. 13E): cleft, 1.1 times as long as wide, each lobe with two distal spines accompanied by long setae, three or one basolateral spines and short setae on dorsal surface. Oostegites (Fig. 12G J): oostegite of gnathopod 2 broad, with long marginal setae, oostegite of pereopods 3 and 4 elongate, oostegite of pereopod 5 smallest. Habitat: Gammarus clarus sp. nov. was found in the source of Jumahe River and along the river, also occurred along Sangan River. All collection sites were at the banks of small streams. Remarks: Gammarus clarus is closely related to G. nekkensis in body shape. Gammarus clarus differs from G. nekkensis (states in parentheses) in inner ramus reaching 0.7 times the length of outer ramus (0.5), both rami fringed with simple and plumose setae (simple setae on outer margin of outer ramus); pereopod 3 with straight setae (curled setae); urosomites 1 2 elevated (flat) The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

40 238 Z. HOU and S. LI GAMMARUS HYPOLITHICUS SP. NOV. (FIGS 14 18) Synonymy: Gammarus sp3 Hou et al., 2007: 599. Material examined: Holotype (IZCAS-I-A317), male, Baishishan Mt. (39 12 N, E), altitude 2096 m, Laiyuan County, Hebei Province, collected by Z. Hou and Y. Lin, 5.ix Paratypes (from IZCAS-I- A317-2 to IZCAS-I-A317-24): 15 males, eight females, same locality with holotype. Etymology: The epithet of the species name is from the habitat, where the new species was found under stone. Diagnosis: Pereopods 3 and 4 with long setae on posterior margins; pereopods 5 7 slender; epimeral plates 1 3 subacute in posterodistal corners; urosomites 1 3 flat; inner ramus of uropod 3 about 0.6 times the length of outer ramus, both rami of uropod 3 with plumose setae; telson with basolateral spines and long setae. Description Holotype: male Body length: 12.3 mm. Head (Fig. 14A): eyes reniform, 1.6 times as long as wide; inferior antennal sinus distinct, ventral recessed margin weakly curving. Antenna 1 (Fig. 14B): peduncular articles 1 3 in length ratio 1 : 0.7 : 0.4, with short lateral and distal setae; flagellum with 30 articles, aesthetascs present from article 4 26; accessory flagellum with five articles; both primary and accessory flagella with short distal setae. Antenna 2 (Fig. 14C): peduncular article 4 about as long as article 5, both with three to four groups of setae along anterior and posterior margins, setae shorter than the width of peduncles; flagellum with 14 articles, calceoli present in article 1 7. Upper lip (Fig. 14D): convex, with minute setae. Left mandible (Fig. 14E): incisor with five teeth; lacinia mobilis with four teeth; spine row with seven pairs of setae; molar with one plumose seta; palp article 2 with 14 marginal setae, article 3 about 0.7 times as long as article 2, with five A-setae, five B-setae, a row of 17 D-setae, and five E-setae. Right mandible (Fig. 14F): incisor with 4 teeth, lacinia mobilis bifurcate, with small teeth. Lower lip (Fig. 14G): inner lobe absent. Maxilla 1 (Fig. 14H, I): asymmetrical, left inner plate with a row of 14 plumose setae; outer plate with 11 serrated apical spines, each spine with small teeth; second article of palp with eight slender spines and four stiff setae on medial surface; right palp wider, article 2 with five stout spines, one pectinate spine, and one seta. Maxilla 2 (Fig. 14J): inner plate with a row of 15 plumose setae and many setules on medial surface; outer plate with stiff apical setae and setules on lateral margin. Maxilliped (Fig. 14K, L): inner plate with three stout apical and one subapical spines; outer plate with 14 blade spines, eight pectinate spines; palp article 4 hooked, with a group of setae at hinge of unguis. Gnathopod 1 (Fig. 15A, C): coxal plate subrectangular, with two setae on anterior corner and one seta on posterior corner; basis with long setae on anterior and posterior margins; carpus short, 1.1 times as long as wide, about 0.6 times as long as propodus; propodus oval, palm oblique, with one medial spine, 17 spines on posterior margin; dactylus with one seta on outer margin and two short setae at hinge of unguis. Gnathopod 2 (Fig. 15B, D): coxal plate with three setae on anterior corner and one seta on posterior corner; basis with long setae along anteroproximal and posterior margins; carpus with parallel margins, 1.6 times as long as wide, about 0.7 times as long as propodus; propodus subrectangular, palm margin subacute, with one medial spine, two spines on medial posterodistal corner, and two spines on lateral posterodistal corner; dactylus with two setae near the hinge of unguis. Pereopod 3 (Fig. 16A, F): coxal plate with three setae on anterior corner and one seta on posterior corner; basis with long setae on posterior margin; merus densely set with long and weakly curled setae on posterior margin, merus with spines accompanied by setae on anterior margin, carpus and propodus with spines and long straight setae on posterior margin; dactylus with one plumose seta on posterior margin and two setae at hinge of unguis. Pereopod 4 (Fig. 16B, G): coxal plate with three setae on anterior corner and eight setae on posterior margin, posterior margin excavated; basis with groups of long setae on posterior margin; merus to propodus with straight setae on posterior margin, carpus and propodus with spines on posterior margin; dactylus with one plumose seta on posterior margin and two stiff setae at hinge of unguis. Pereopods 5 7 slender. Pereopod 5 (Fig. 16C, H): coxal plate with one seta on anterior corner and two setae on posterodistal corner; basis posterior margin straight, posterior corner subquadrate, with one long seta and five spines on anterior margin and a row of nine setae on posterior margin; merus to propodus with spines along anterior and posterior margins accompanied by 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

41 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 239 Figure 14. Gammarus hypolithicus sp. nov., male. A, head; B, antenna 1; C, antenna 2; D, upper lip; E, left mandible; F, incisor of right mandible; G, lower lip; H, left maxilla 1; I, palp of right maxilla 1; J, maxilla 2; K, maxilliped; L, dactylus of palp of maxilliped The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

42 240 Z. HOU and S. LI Figure 15. Gammarus hypolithicus sp. nov., male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2; E, epimeral plate 1; F, epimeral plate 2; G, epimeral plate 3; H, telson The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

43 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 241 Figure 16. Gammarus hypolithicus sp. nov., male. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, dactylus of pereopod 3; G, dactylus of pereopod 4; H, dactylus of pereopod 5; I, dactylus of pereopod 6; J, dactylus of pereopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

44 242 Z. HOU and S. LI Figure 17. Gammarus hypolithicus sp. nov., male, A G; female, H, I. A, pleopod 1; B, pleopod 2; C, pleopod 3; D, uropod 1; E, uropod 2; F, uropod 3; G, urosomites 1 3; H, uropod 3, I, telson The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

45 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 243 Figure 18. Gammarus hypolithicus sp. nov., female. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2; E, oostegite of gnathopod 2; F, oostegite of pereopod 3; G, oostegite of pereopod 4; H, oostegite of pereopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

46 244 Z. HOU and S. LI few short setae; dactylus with one seta on posterior margin and two setae at hinge of unguis. Pereopod 6 (Fig. 16D, I): coxal plate with three setae on posterodistal corner; basis elongate, with two groups of long setae and four spines on anterior margin, posterior margin proximally broad, distally narrow, with a row of 13 setae, inner surface with one seta; merus to propodus with groups of spines accompanied by short setae along anterior and posterior margins; dactylus with one plumose seta on posterior margin and one seta at hinge of unguis. Pereopod 7 (Fig. 16E, J): coxal plate with two setae on anterior margin and four setae on posterior margin; basis with four long setae and a row of five spines on anterior margin, posterior margin expanded, shape convex, with 15 setae on posterior margin, inner surface with one spine accompanied by two setae; merus to propodus with two to four groups of spines accompanied by few short setae on anterior margin, and propodus with two groups of setae on posterior margin; dactylus with one plumose seta on posterior margin and two stiff setae at hinge of unguis. Coxal gills: coxal gill of gnathopod 2 and gills of pereopods 3 5 a little shorter than bases; gill of pereopod 6 about half length of basis; gill of pereopod 7 smallest, less than half of basis. Epimeral plates (Fig. 15E G): epimeron 1 ventrally rounded, with eight long setae and two stiff setae on anterior corner, and three setae on posterior margin; epimeron 2 with one long seta on anterior corner, one subventral spine and two spines on ventral margin, posterodistal corner weakly produced, subacute, with five setae on posterior margin; epimeron 3 with four long setae on anterior corner, three spines on ventral margin, posterodistal corner weakly produced, subacute, with three setae on posterior margin. Pleopods 1 3 subequal (Fig. 17A C), peduncle with long facial and marginal setae, with two retinacula accompanied by one to two setae on anterodistal corner; both inner and outer rami fringed with plumose setae. Urosomites 1 3 dorsally flat (Fig. 17G), urosomite 1 with spine with short setae on dorsal margin; urosomite 2 with spines accompanied by short setae on dorsal margin; urosomite 3 with two spines accompanied by setae on each side and one or no seta on dorsal margin. Uropod 1 (Fig. 17D): peduncle with one basofacial spine, three spines on outer margin, one spine on inner margin, with one and two spines on inner and outer distal corners, respectively; outer ramus with one spine on outer margin and one spine on inner margin; inner ramus with one spine on inner margin, both rami with five distal spines. Uropod 2 (Fig. 17E): peduncle with two and one spines on outer and inner margin, respectively, one spine on inner and outer corner, respectively; outer and inner rami with one spine on outer and inner margin, respectively. Uropod 3 (Fig. 17F): peduncle with one lateral spine and six distal spines; inner ramus 1.4 times the length of peduncle, reaching 0.6 times the length of outer ramus, with two spines on lateral margin and two distal spines; article 1 of outer ramus with one single spine and three pairs of spines on outer margin and two pairs of spines on distal margin, both margins densely set with plumose and simple setae, terminal article as long as adjacent spines. Telson (Fig. 15H): deeply cleft, 0.9 times as long as wide, each lobe with two distal spines accompanied by long setae, one basolateral spine, and long setae on lateral surface. Female, paratype (IZCAS-I-A317-2) Body length: 10.3 mm. Gnathopod 1 (Fig. 18A, C): coxal plate with two setae on anterior corner and one seta on posterior margin; propodus oval, palm margin not as oblique as that of male, with eight spines on posterodistal corner; dactylus with one seta on outer margin and two setae at hinge of unguis. Gnathopod 2 (Fig. 18B, D): propodus subrectangular, palm margin transverse, with five spines on posterodistal corner; dactylus with one seta on outer margin and one seta at hinge of unguis. Pereopods 3 and 4 with straight setae on posterior margin. Uropod 3 (Fig. 17H): inner ramus about 0.6 times as long as outer ramus, both rami with plumose setae, terminal article of outer ramus as long as adjacent spines. Telson (Fig. 17I): cleft, 0.8 times as long as wide, each lobe with two distal spines accompanied by long setae, one basolateral spine, and long setae on dorsal surface. Oostegites (Fig. 18E H): oostegite of gnathopod 2 broad, with long marginal setae, oostegite of pereopods 3 and 4 elongate, oostegite of pereopod 5 smallest. Habitat: Gammarus hypolithicus sp. nov. was only found in the type locality, a small pool near the peak of Baishishan Mt. This pool was less than 1 m 2,and water fell from a height of 10 m. Remarks: Gammarus hypolithicus sp. nov. differs from G. nekkensis (state in parentheses) in long straight setae on posterior margin of pereopods 3 and 4 (curled setae), both margins of rami of uropod 3 with plumose setae (outer margin of outer ramus with simple setae). Gammarus hypolithicus sp. nov. is dis The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

47 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 245 tinguished from G. clarus (state in parentheses) by epimeral plate 3 with small acute tooth (acute); urosomites 1 2 flat, with four groups of spines and setae on dorsal margin (elevated, with three groups of spines and setae on dorsal margin); telson with long setae (short setae). GAMMARUS PARVIOCULUS SP. NOV. (FIGS 19 23) Synonymy: Gammarus sp4 Hou et al., 2007: 599. Material examined: Holotype (IZCAS-I-A318), male, Tongdong Cave (39 42 N, E), Sihe Village, Xiayunling Town, Fangshan District, Beijing City; collected by X. Xu and Q. Wang, 13.iv Paratypes (from IZCAS-I-A318-2 to IZCAS-I-A318-23), 16 males and six females, same locality as holotype. Etymology: The specific name is from the Latin parvi- and -oculus, referring to the vestigial eyes. Diagnosis: Eyes small; pereopods 3 7 slender, with few long setae on anterior and posterior margins; inner ramus less than half of outer ramus, outer margin of outer ramus with simple setae. Description Holotype: male Body length: 12.5 mm. Head (Fig. 19A): eyes relatively small, about onesixth of head depth, 1.2 times as long as wide; inferior antennal sinus distinct, projection on the anterodistal corner shorter. Antenna 1 slender (Fig. 19B), peduncular articles 1 3 in length ratio 1 : 0.8 : 0.4, with distal setae; flagellum with 32 articles, most with aesthetascs; accessory flagellum with five articles. Antenna 2 (Fig. 19C): peduncular article 5 a little longer than article 4, with groups of short setae along anterior and posterior margins; flagellum with 14 articles, calceoli present. Upper lip subrounded (Fig. 19D), with minute setae. Mandibles (Fig. 19F): left incisor with five teeth; lacinia mobilis with four teeth; spine row with eight pairs of plumose setae; article 2 of palp with 12 marginal setae, article 3 with a group of four A-setae, a group of three B-setae, a row of plumose D-setae, and five E-setae. Right incisor with four teeth (Fig. 19E); lacinia mobilis bifurcate, with dentitions at edge. Lower lip (Fig. 19G): inner plate absent. Maxilla 1 asymmetric (Fig. 19H, I, M), inner plate with a row of 14 plumose setae; outer plate with 11 serrated spines; article 2 of left palp with seven slender spines accompanied by three stiff setae; article 2 of right palp with five stout spines and one slender spine accompanied by one seta. Maxilla 2 (Fig. 19J): inner plate with a diagonal row of 15 plumose setae; outer plate with apical setae. Maxilliped (Fig. 19K, L): inner plate with three apical spines and one to two subapical spines; outer plate with a row of 15 spines on inner margin and six apical pectinate spines; palp with four articles. Gnathopod 1 (Fig. 20A, C): coxal plate with two and one setae on anterior and posterior corners, respectively, lower margin with setules; basis with long setae on anterior and posterior margins; carpus 1.6 times as long as wide, about 0.75 times as long as propodus; propodus ovate, with one stout spine on medial palmar margin, posterior margin with one single and four pairs of spines, inner surface with three groups of three, three, and two spines; dactylus with one seta on outer margin. Gnathopod 2 (Fig. 20B, D, E): coxal plate with three setae on anterior corner and one seta on posterior corner; carpus with parallel sides, 1.6 times as long as wide, about 0.83 times as long as propodus; propodus palm subacute, with one stout medial palmar spine and two pairs of spines on posterodistal corner, inner surface with groups of long setae; dactylus with one seta on outer margin and several short setae at hinge of unguis. Pereopod 3 (Fig. 21A, F): coxal plate subrectangular, with two setae on lower margin; basis with long setae on posterior margin; merus and carpus densely set with long and straight setae on posterior margin; propodus with long setae accompanied by spines; dactylus with one plumose seta on outer margin and two setae at hinge of unguis. Pereopod 4 (Fig. 21B, G): coxal plate excavated on posterior margin, with setae on anterior corner and posterior margin; basis with long setae on posterior margin; merus to propodus with groups of setae on posterior margin accompanied by spines; dactylus with one plumose seta on posterior margin and two setae at hinge of unguis. Pereopod 5 (Figs 21C, 22A): coxal plate with one seta on anterior corner and two setae on posterior margin; basis with six single spines on anterior margin, posterior margin straight with a row of short setae, posterior corner subquadrate; merus and carpus with two groups of spines on anterior and posterior margins; propodus with three groups of spines on anterior margin; dactylus with one plumose seta on outer margin and two stiff setae at hinge of unguis. Pereopod 6 (Figs 21D, 22B): slender, basis elongate, posterior margin weakly expanded, inner surface with one seta; merus to propodus with spines and few short setae, carpus longer than merus and propodus; 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

48 246 Z. HOU and S. LI Figure 19. Gammarus parvioculus sp. nov., male. A, head; B, antenna 1; C, antenna 2; D, upper lip; E, right incisor and lacinia mobilis; F, left mandible; G, lower lip; H, left maxilla 1; I, outer plate of left maxilla 1; J, maxilla 2; K, maxilliped; L, inner plate of maxilliped; M, palp of right maxilla The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

49 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 247 Figure 20. Gammarus parvioculus sp. nov., male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1 (inner surface); D, propodus of gnathopod 2 (inner surface); E, propodus of gnathopod 2 (outer surface) The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

50 248 Z. HOU and S. LI Figure 21. Gammarus parvioculus sp. nov., male. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, dactylus of pereopod 3; G, dactylus of pereopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

51 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 249 Figure 22. Gammarus parvioculus sp. nov., male. A, dactylus of pereopod 5; B, dactylus of pereopod 6; C, dactylus of pereopod 7; D, epimeral plate 1; E, epimeral plate 2; F, epimeral plate 3; G, pleopod 1; H, uropod 1; I, uropod 2; J, uropod 3; K, telson The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

52 250 Z. HOU and S. LI Figure 23. Gammarus parvioculus sp. nov., female. A, propodus of gnathopod 1; B, propodus of gnathopod 2; C, telson; D, telson; E, oostegite of gnathopod 2; F, oostegite of pereopod 3; G, oostegite of pereopod 4; H, oostegite of pereopod The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

53 DELIMITATION OF GAMMARUS SPECIES BOUNDARIES 251 dactylus with one plumose seta on outer margin and two stiff setae at hinge of unguis. Pereopod 7 slender (Figs 21E, 22C), coxal plate with five setae on posterior margin; posterior margin of basis weakly sinuate, inner surface with one spine accompanied by one seta; merus to carpus similar to those of pereopod 6. Coxal gills present on gnathopod 2 and pereopods 3 7. Epimeral plates (Fig. 22D F): epimeron 1 with ten long setae on anteroventral corner, posterior margin with four setules; epimeron 2 posterior margin nearly straight, bearing two setae and three spines on ventral margin and five setae on posterior margin; epimeron 3 posterodistal corner weakly produced, bearing four long setae on anterior margin, three spines on ventral margin, and three setae on posterior margin. Pleopods 1 3 subequal (Fig. 22G), peduncle with two retinacula accompanied by one to three plumose setae on anterior corner; outer ramus a little shorter than inner ramus, both rami fringed with plumose setae. Urosomites 1 3 dorsally flat, with four groups of spines and setae. Uropod 1 (Fig. 22H): peduncle with one basofacial spine, outer margin with three spines and inner margin with one spine; inner ramus with one spine on inner margin; outer ramus with one spine on inner and outer margins, both rami with five distal spines. Uropod 2 (Fig. 22I): peduncle longer than both rami, with two spines on outer margin and one spine on inner margin; outer ramus with one spine on inner and outer margins; inner ramus with one spine on inner margin. Uropod 3 (Fig. 22J): peduncle with one spine accompanied by one seta on lateral surface; inner ramus nearly the same length as peduncle, about 0.4 times as long as outer ramus, inner and outer margins with plumose and simple setae; article 1 of outer ramus with two pairs of spines and long simple setae on outer margin, inner margin densely set with long simple and plumose setae on inner margin, terminal article shorter than adjacent spines. Telson cleft (Fig. 22K), length same as width, each lobe with one basolateral spine accompanied by long facial setae, with two distal spines accompanied by long setae. Female: paratype (IZCAS-I-A318-2) Body length: 9 mm. Gnathopod 1 (Fig. 23A): propodus oval, palm not as oblique as that of male, without palmar medial spine, with four spines on posterodistal corner. Gnathopod 2 (Fig. 23B): propodus longer than that of male, with three spines on posterodistal corner. Uropod 3 (Fig. 23C): inner ramus about 0.44 times as long as outer ramus; outer ramus with two pairs of spines and long simple setae on outer margin, inner margin with simple and plumose setae, terminal article shorter than adjacent spines; inner ramus with simple and plumose setae on both margins. Telson cleft (Fig. 23D), 0.8 times as long as wide, each lobe with basofacial and distal spines accompanied by long setae. Oostegites 2 5 progressively reduced (Fig. 23E H), with long marginal setae. Habitat: The examined specimens were collected 15 m away from the entrance of cave. This entrance is halfway up the hill, and there is a crystal-clear underground river running throughout the year. Remarks: Gammarus parvioculus sp. nov. is very similar to G. nekkensis in antenna 2 with calceoli, epimeral plates 2 and 3 posterodistal corners weakly produced, and uropod 3 with long simple setae on outer margin of outer ramus. Gammarus parvioculus differs from G. nekkensis (state in parentheses) in smaller eyes, about one-sixth of head (one-quarter), pereopod 3 with long, straight setae on posterior margin (curled setae), pereopods 5 7 very slender, inner ramus of uropod 3 about 0.4 times the length of outer ramus (0.5 times as long as outer ramus). The comparisons with the other three new species are presented in Table 2. This taxon may have adapted to a cave-entrance existence relatively recently and therefore is intermediate between a common freshwater species with large eyes and a cave species without eyes. ACKNOWLEDGEMENTS We would like to thank J. Fu of the University of Guelph, and S. Menken and D. Platvoet of the University of Amsterdam for reading this manuscript and offering valuable comments. We also thank P. Hayward of the University of Wales Swansea, A.-N. Lörz of the National Institute of Water and Atmospheric Research (NIWA), New Zealand, and an anonymous reviewer for a number of useful comments and suggestions. This study was supported by the National Natural Sciences Foundation of China (NSFC / / / ), by the National Science Fund for Fostering Talents in Basic Research (Special Subjects in Animal Taxonomy, NSFC-J /J0109), by the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-Z-008/KSCX3-IOZ-0811), by the Ministry of Science and Technology of the People s Republic of China (MOST grant no. 2006FY120100/ 2006FY110500), and partly also by the Beijing Natural Science Foundation ( ) The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

54 252 Z. HOU and S. LI Table 2. Comparisons of morphological characters separating the four new species Species name Armature on dorsal margin of urosomite 1 Ratio of inner ramus and outer ramus of uropod 3, and setae Eyes Posterodistal corner of epimeral plate 3 Gammarus illustris sp. nov. Gammarus clarus sp. nov. Gammarus hypolithicus sp. nov. Gammarus parvioculus sp. nov. Flat, with four groups of setae and no spines Elevated, with three groups of spines and setae Flat, with four groups of spines and setae Flat, with four groups of spines and setae 0.2, with simple setae on both margin 0.7, with plumose and simple setae on both margin 0.6, with plumose and simple setae on both margins 0.4, with simple setae on outer margin of outer ramus, plumose and simple on inner margin Medium, 1.6 times as long as wide Medium, 2.2 times as long as wide Medium, 1.6 times as long as wide Small, 1.2 times as long as wide With small acute tooth Acute With small acute tooth With small acute tooth REFERENCES Barnard JL, Barnard CM Freshwater Amphipoda of the world I, II. Mt. Vernon, VA: Hayfield Associates. Cracraft J Species concepts and speciation analysis. In: Johnston RF, ed. Current ornithology, Vol. 1. New York: Plenum Press, Fabricius JC Systema Entomologiae sistens Insectorum classes, ordines, genera, species adjectis synonymis, loci, descriptionibus, observationibus. Flensburg and Leipzig. Felsenstein J Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: Folmer O, Black M, Hoeh W, Vrijenhoek R DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: Funk DJ, Omland KE Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annual Review of Ecology, Evolution and Systematics 34: Guralnick RP, Hill AW, Lane M Towards a collaborative, global infrastructure for biodiversity assessment. Ecology Letters 10: Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM Identification of birds through DNA barcodes. Public Library of Science Biology 2: Hillis DM, Mable BK, Larson A, Davis SK, Zimmer EA Nucleic acids IV: sequencing and cloning. In: Hillis DM, Moritz C, Mable BK, eds. Molecular systematics, 2nd edn. Sunderland, MA: Sinauer Associates, Holsinger JR Systematics, speciation, and distribution of the subterranean amphipod genus Stygonectes (Gammaridae). Bulletin of the United States National Museum 259: Hou Z Systematics of Chinese freshwater Amphipoda. PhD dissertation, Graduate University of Chinese Academy of Sciences, Beijing. Hou Z, Fu J, Li S A molecular phylogeny of the genus Gammarus (Crustacea: Amphipoda) based on mitochondrial and nuclear genes. Molecular Phylogenetics and Evolution 45: Hou Z, Li S Two new freshwater gammarids (Crustacea: Amphipoda: Gammaridae) from Lake Lugu, China. Revue suisse de Zoologie 110: International Commission on Zoological Nomenclature International code of zoological nomenclature, 4th edn. London: The International Trust for Zoological Nomenclature. Karaman GS One freshwater Gammarus species (Gammaridea, Fam. Gammaridae) from China (Contribution to the knowledge of the Amphipoda 1989). Poljoprivreda I Sumarstvo 35: Karaman GS, Pinkster S. 1977a. Freshwater Gammarus species from Europe, North Africa and adjacent regions of Asia (Crustacea-Amphipoda). Part I. Gammarus pulexgroup and related species. Bijdragen tot de Dierkunde, Amsterdam 47: Karaman GS, Pinkster S. 1977b. Freshwater Gammarus species from Europe, North Africa and adjacent regions of Asia (Crustacea-Amphipoda). Part II. Gammarus roeseligroup and related species. Bijdragen tot de Dierkunde, Amsterdam 47: Karaman GS, Pinkster S Freshwater Gammarus species from Europe, North Africa and adjacent regions of Asia (Crustacea-Amphipoda). Part III. Gammarus 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160,

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56 Hydrobiologia (2010) 649: DOI /s PRIMARY RESEARCH PAPER Is Gammarus tigrinus (Crustacea, Amphipoda) becoming cosmopolitan through shipping? Predicting its potential invasive range using ecological niche modeling Jiawen Ba Zhonge Hou Dirk Platvoet Li Zhu Shuqiang Li Received: 17 November 2009 / Revised: 11 March 2010 / Accepted: 22 March 2010 / Published online: 5 April 2010 Ó Springer Science+Business Media B.V Abstract While the intensity of global shipping has increased dramatically over the last decades, species exchange between continents has likewise intensified. Ballast water of ships is recognized playing a major role in this process. Many of the larger sea ports have become bridgeheads for invasions. Ecological niche modeling is used to investigate the potential invasive range and high invasive risk ports of the North American amphipod Gammarus tigrinus. Sixty-two occurrences of G. tigrinus in its native range (North America) and 34 environmental data sets were compiled. Data on dispersal distances were used via ecological niche modeling to analyze the invasive Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. Handling editor: T. P. Crowe J. Ba Z. Hou S. Li (&) Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing , People s Republic of China lisq@ioz.ac.cn D. Platvoet Zoological Museum of the University of Amsterdam, Mauritskade 57, 1092 AD Amsterdam, The Netherlands L. Zhu Institute of Botany, Chinese Academy of Sciences, Beijing , China potential of G. tigrinus. The invasive risk of large ports was analyzed according to modeling result, as well as their salinity in the main oceanic routes of the world. G. tigrinus had a rapid range extension on the British Isles and in the rest of Western Europe. Now it is invading the countries surrounding the Baltic Sea. Worldwide it has a vast potential invasive range. It has a high invasive risk for many large ports along the main oceanic routes, among which the ports of Shanghai, Buenos Aires and Montevideo have the highest invasive risk. G. tigrinus may become cosmopolitan through shipping, and this possibility is increasing. Particular emphasis should be placed on preventing human-mediated dispersal. Ports may be the first places G. tigrinus invades. This study can identify high invasive risk ports, especially those at risk of introduced North America species. More importantly, the water of large ports should be monitored regularly for exotic aquatic organisms that may survive temporarily or permanently. Keywords Ballast water Amphipods Invasive range Human-mediated dispersal Introduction Transportation is increasingly being recognized as the major species dispersion vector. Globalization of international trade has promoted numerous biological invasions and the rate at which species are being 123

57 184 Hydrobiologia (2010) 649: transported is unprecedented (Levine & D Antonio, 2003; Perrings et al., 2005). Around 7,000 marine and coastal species travel across the world s oceans every day (Battle, 2009). Eighty-four percent of the world s marine regions reported invasive species in 2008, with shipping being a major reason for their spread ( Invasive marine and freshwater species are being spread around the world in ship s ballast water, damaging industries and natural resources (Dunstan & Bax, 2008). Ballast water is probably the most important mechanism where aquatic invasions are concerned (Cohen, 1998). Although many organisms cannot survive the dark and often dirty conditions in ballast tanks for a long period, the increased speed of modern vessels has improved the survival rate of species and individuals in transoceanic transports (Costello et al., 2007). For an invasive species a matching habitat may facilitate a successful invasion (Stohlgren et al., 2006). Animals must obey the same ecological rules in invaded ranges as they do in native ranges, and knowledge of the ecological characteristics of native ranges may give indications for potential dispersion localities. A sizeable body of evidence is accumulating to support the idea that, at least on a coarse spatial scale, ecological shifts rarely accompany species invasions (Richardson & McMahon, 1992; Higgins et al., 1999; Iguchi et al., 2004; Robertson et al., 2004). However, accurate prediction of future species distributions is challenging. This requires knowledge of the number of individuals introduced into a particular area over time (i.e., propagule pressure), as well as measures of environmental suitability. Only if both these criteria are met, can successful invasions take place (Herborg et al., 2007a). The Genetic Algorithm for Rule-set Prediction (GARP) (Stockwell & Noble, 1992; Stockwell & Peters, 1999) is a system that has excellent capabilities for delineating ecological niches and predicting the geographic distributions of species. GARP has been utilized to predict potential distributions of invasive species, such as Zebra Mussels (Dreissena polymorpha) in the western USA (Drake & Bossenbroek, 2004), Chinese mitten crabs (Eriocheir sinensis) in Europe and USA (Herborg et al., 2007a, b), Anoplophora glabripennis in North America (Peterson & Pereira, 2004), Barred Owl in North America (Peterson & Robins, 2003), and Quadrastichus erythrinaw worldwide (Li et al., 2006). The gammarid Gammarus tigrinus Sexton, 1939 is a widespread species that occurs principally in estuaries of the northwestern Atlantic and is distributed from the St. Lawrence River in Quebec to Florida (Bousfield, 1958, 1973). It is dominant in intertidal and subtidal benthic habitats including reeds, algae, hard or soft substratum, and sand (Bousfield, 1958, 1973; van Maren, 1978). In its native range, this euryhaline species lives in both fresh and brackish water (Bousfield, 1973), whereas in coastal waters it is restricted to shallow lagoons, bays, and estuaries. Being a bottom dwelling omnivore, G. tigrinus is relatively tolerant to pollution and has a much greater reproductive capacity in oligohaline waters than many native gammarid species (Pinkster, 1975; Pinkster et al., 1977). The species is able to form populations in a wide range of inland and coastal ecosystems in temperate regions. Unlike some epiphytic amphipods, G. tigrinus is unlikely to disperse by algal rafting (Myers, 1993). Its distribution showed a continuous rather than fragmented pattern. There is a high risk of further expansions to the various lakes of Eastern Europe via inland canal river systems, which may lead to unforeseeable changes in aquatic communities (Berezina, 2007). It was introduced into British waters by ballast water in 1931 (Sexton & Cooper, 1939), then spread to similar habitats in Western Europe and, most recently, to Eastern Europe, the Baltic Sea, and the Laurentian Great Lakes (Kelly et al., 2006b). The spread of this amphipod in European continental waters was accelerated by its intentional release into the tributary of the German River Weser in 1957 (Schmitz, 1960). In the Netherlands, this species was first recorded from the IJsselmeer in 1964 (Pinkster, 1975) and in 1975 reached the south-western part of the Baltic Sea (Schlei Fjord) probably via the Nord- Ostsee-Kanal (Bulnheim, 1976). After two decades, its rapid spread in German waters along the southern Baltic coastline was noticed (Rudolph, 1994; Zettler, 1995) and soon the species was found in Szczecin Lagoon (Gruszka, 1995), Puck Bay (Gruszka, 2002) and the Vistula Lagoon (Ja_zd_zewski & Konopacka, 2000). The latter water body was the northern limit of the species in the Baltic until its occurrence in the Gulf of Riga (Kotta, 2005) and along the Finnish coast in 2003 (harbors of Hamina in the Gulf of Finland and Turku in the northern Baltic) (Pienimäki et al., 2004). Gammarus tigrinus was discovered in 123

58 Hydrobiologia (2010) 649: the Lithuanian part of Curonian Lagoon in September 2004 (Daunys & Zettler, 2006). In 2006, it was found in the easternmost part of Baltic Sea (Neva Estuary) and may well have been transported there with ballast waters from the Finnish area of the Gulf of Finland (Berezina, 2007). Dispersion in the last decade was extremely rapid in Eastern Europe. In South America, it was found in the Gulf of Paria, and the Orinoco Delta, Venezuela (Capelo et al., 2004; Martín & Díaz, 2007). The initial introduction of G. tigrinus into Europe via ballast water (Sexton & Cooper, 1939) confirmed its survival during transportation in ballast tanks. The comparison of life history traits indicated that G. tigrinus has a high tolerance toward salinity changes (0 25 PSU) and human degradation of the environment and is well adapted to withstand predation (Grabowski et al., 2007a, b). Concurrent with the invasion of G. tigrinus, the diversity of native gammarids has declined (Herkül & Kotta 2007). Gammarus tigirnus is not only restricted to near port water bodies, but is also widespread in inland water systems. At present, it is difficult to foresee natural pressures which may limit the spread or establishment of the alien G. tigrinus. The global shipping industry has developed rapidly, and most of the maritime traffic (85%) takes place in the northern hemisphere (Corbett et al., 1999; Endresen et al., 2003). The international sea borne cargo statistics indicate that the USA, Europe and East Asia are the main export regions of ballast water by crude oil carriers. The pattern is different for bulk vessels where the most important export areas of ballast water are Asia and Europe while the importing regions are North and South America, Australia and Asia. In the present study, we have collected the distribution data of G. tigrinus in both native ranges and introduced ranges. On the basis of the distribution data, we used the GARP models to predict the potential distribution of G. tigrinus and to identify areas most vulnerable to future invasion. Predicting future ranges of nonindigenous species using only environmental niche models may provide misleading forecasts since many areas suitable for colonization may lack appropriate vectors to transmit the species to these locations (Herborg et al., 2007a). In particular, we combined the result of ecological niche modeling (ENM) and salinity for some larger ports in the potential invasive regions and the main ocean routes of the world to analyze the invasive potential of G. tigrinus. Methods Species data We compiled a database of the distribution of G. tigrinus in its native range (North America) and non-native range (Europe and Venezuela) from the Global Biodiversity Information Facility (Anonymity, 2009) and papers (Daunys & Zettler, 2006; Kelly et al., 2006a, b; Berezina, 2007; Martín&Díaz, 2007; Piscart et al., 2007; Arbačiauskas, 2008). In the Netherlands, it had a large number of records for G. tigrinus, so we only selected some localities at random. The detailed invasive distribution and history of G. tigrinus were listed in Table 1. A total of 127 distinct, spatially unique locations that had at least 1 min difference in coordinates (Levine & D Antonio, 2003) were identified (Appendix 1). Distributional records were available from the UK, Germany, Northern Ireland, the Netherlands, Poland, Russia, Finland, France, Estonia, Latvia, and Lithuania. Records with unspecified or unknown localities were not considered; the remaining records were plotted on maps and inspected visually to detect obvious errors using ArcMap (9.0). For ENM analyses, occurrence locations were considered only once, with no weighting to account for multiple cases at single locations. Geographic data Environmental data sets input into GARP came from three principal sources. (1) January and July average maximum and minimum temperature, January and July average precipitation and 19 other factors related to bioclimatic data were obtained from global land area interpolation of climate point data ( ) at a spatial resolution of 2.5 arc-min ( worldclim.org) (Table 2, No. 1 25); (2) Other climatic data averaged over the period , including data layer summarizing wet days, water vapor pressure and solar radiation were drawn from the Intergovernmental Panel on Climate Change ( (Table 2, No ); (3) Landsurface data summarizing elevation, aspect, flow 123

59 186 Hydrobiologia (2010) 649: Table 1 Historical invasion of Gammarus tigrinus in nonnative range (EU Europe, NA North America, SA South America) Continent Country Water body First report Transport vector Source NA Canada Laurentian Great Lakes 2002 Kelly et al. (2006b) EU Estonian Kõiguste Bay 2003 Herkül & Kotta (2007) EU Finland Gulf of Finland 2003 Ballast water Pienimäki et al. (2004) EU Finland Turku 2003 Ballast water Pienimäki et al. (2004) EU France Brittany Piscart et al. (2007) EU France Meurthe River 2002 Piscart et al. (2005) EU France Moselle River 1999 Devin et al. (2001) EU Germany Schlei estuary 1975 Intentional release Bulnheim (1976) EU Germany Mecklenburg area 1994 Intentional release Rudolph (1994) EU Germany Weser River 1957 Intentional release Schmitz (1960) EU Ireland Lough Neagh; Bann River 1931 Ballast water Sexton & Cooper (1939) EU Latvia Gulf of Riga 2003 Kotta (2005) EU Lithuanian Curonian Lagoon 2004 Daunys & Zettler (2006) EU Luxembourg Moselle (Germany-Luxembourg 1991 Intentional release Massard & Gaby (1992) border near D-Nennig) EU The Netherlands Ijsselmeer 1960 Intentional release Nijssen & Stock (1966) EU Poland Szczecin Lagoon 1988 Ballast water Gruszka (1995) EU Poland Puck Bay 2002 Szaniawska et al. (2003) EU Poland Vistula Lagoon 1998 Ballast water Ja_zd_zewski & Konopacka (2000) EU Russia Neva Estuary 2006 Ballast water Berezina (2007) EU Russia Kaliningrad province Ezhova et al. (2005) EU England Frodsham Marsh 1931 Ballast water Sexton & Cooper (1939) SA Venezuela Orinoco Delta; Gulf of Paria Ballast water Capelo et al. (2004) accumulation, flow direction, slope, and topographic index were obtained from the U.S. Geological Survey s Hydro-1K data set ( gov/gtopo30/hydro) (Table 2, No ). All 34 environmental data sets were resampled to 0.1 for analysis to match the approximate resolution of occurrence data. Ecological niche modeling Ecological niches and potential geographic distributions were modeled using the GARP ( lifemapper.org/desktopgarp). GARP is an application that builds ENM based on nonrandom associations between known occurrence points for species and sets of raster GIS coverages describing ecological landscapes (Gaubert et al., 2006). It is a widely applied environmental niche modeling application that uses raster-based environmental and biological information to predict a suitable habitat for a given species (Herborg et al., 2007a). In order to reduce environmental coverage sets to just those coverages that provide highest predictive accuracy, we used a jackknife manipulation and analysis to test the effect of different environmental layers on prediction precision. Sets of geographical coverages were reduced to an optimal 24 ecological dimensions in the final model (Peterson & Cohoon, 1999; Zhu et al., 2007) (Table 2, Variables included in the final analysis is indicated with a H). In our model, we used 62 unique occurrence points from the native distribution of G. tigrinus to develop the algorithm. The GARP model selected nonrandom associations between environmental layers and presence of G. tigrinus in its native range with a genetic algorithm. The algorithm develops a set of conditional 123

60 Hydrobiologia (2010) 649: Table 2 Description of environmental variables in the coverage set Variables included in the final analysis are indicated with a H Description 1 Annual mean temperature 2 Mean diurnal range (mean of monthly (max temp - min temp)) H 3 Isothermality (P2/P7) (*100) H 4 Temperature seasonality (standard deviation * 100) H 5 Max temperature of warmest month 6 Min temperature of coldest month 7 Temperature annual range (P5 P6) 8 Mean temperature of wettest quarter 9 Mean temperature of driest quarter H 10 Mean temperature of warmest quarter H 11 Mean temperature of coldest quarter H 12 Annual precipitation H 13 Precipitation of wettest month 14 Precipitation of driest month H 15 Precipitation seasonality (coefficient of variation) 16 Precipitation of wettest quarter H 17 Precipitation of driest quarter H 18 Precipitation of warmest quarter 19 Precipitation of coldest quarter H 20 January average maximum temperature H 21 January average minimum temperature H 22 July average maximum temperature H 23 July average minimum temperature H 24 January average precipitation H 25 July average precipitation 26 Wet days (number of days of precipitation) H 27 Water vapour pressure 28 Solar radiation H 29 Elevation H 30 Aspect (direction of the slope) H 31 Flow accumulation (the amount of upstream area draining H into each cell, drainage area) 32 Flow direction H 33 Slope (maximum change in elevation between each cell and H its eight neighbours) 34 Topographic index H Included rules from an available range (i.e., atomic rule, logistic regression), then iteratively improves the solution by testing and selecting rules on random subsets of available data (Stockwell & Peters, 1999). Presence data are randomly divided by the GARP program into 80% training and 20% validation data. Models were generated with a maximum of 10,000 iterations and a convergence limit. The final prediction maps were produced by summing these 10 high-quality models (Anderson et al., 2003). This approach selects models with a false negative rate (omission error) of \5% and a false positive rate (commission error) of \50%. The intersection of all the 10 best-subset models generated a final map with values ranging from 0 to 10 (10 for regions where all the predicted niche models present; 0 for regions of niche absence). Color 123

61 188 Hydrobiologia (2010) 649: gradations are used to indicate the proportion of times out of 10 that specific areas were included in the predicted distribution of G. tigrinus. Dispersal limitation The high invasive risk range of the alien freshwater invertebrates through ballast water mainly spread along coastal areas. G. tigrinus is an extremely euryoecious species and characteristic to shallow water habitats (Grabowski et al., 2007a). In order to distinguish the higher invasive potential range, we developed a dispersal distance layer in the ecological niche model (Herborg et al., 2007a). We measured the distance between locations of reported occurrence and the nearest coastline and identified the 90th percentile (306 km) for the distribution of inland dispersal distances in Europe and Venezuela (Fig. 1). The coastline of Europe is rather irregular, the invasive regions are not very far from the coasts. Therefore, our model does not have limiting dispersal distances in Europe. This limit was selected since it is not known whether G. tigrinus were established at the most distant sites for which they are reported in Europe and Venezuela, but they are established at the 90th percentile distance. We applied the 306 km distances as separate cut-off points for maximum expected dispersal distances for G. tigrinus in their native and some potential invasive ranges. The resulting layers identified waterways that are suitable for survival and are within established dispersal distance limits based on the G. tigrinus s native distribution. Fig. 1 Dispersal distances of G. tigrinus in nonnative range. Dispersal distance is calculated as the distance from the freshwater point of occurrence to the nearest coastline. Distances were measured using DIVA-GIS software Ports and ocean route We selected some major ports in the regions within the potential invasive range of G. tigrinus according to our model, because these major ports receive large amounts of ballast water that may carry nonindigenous aquatic invertebrates. We did not consider the ports of Europe, because the area is surrounded by the Atlantic Ocean, the Baltic Sea, the Mediterranean Sea and the Black Sea, possessing abundant ports. These ports have short dispersal distances between them and more importantly they had a longer history of invasions. In addition, we considered the salinity of port water (World Ocean Data 2005, noaa.gov) and the main oceanic routes in the world, because these routes connect larger ports with a high invasion or dispersal potential. Results Potential distribution range From the results of our model it may be concluded that G. tigrinus has a wide potential invasive range worldwide (Fig. 2A). Major areas with potential invasions are the central west coast of North America, the north coast of the Mediterranean Sea, East Asia, the estuary of the River Plate in South America, the regions Rio de Janeiro in Brazil, the coastal areas of Gulf of Guinea in Africa, and the southeast coastal areas of Australia. The main ocean routes play an important role in transmitting exotic aquatic species through ballast water. Most of the maritime traffic (85%) takes place in the northern hemisphere, specifically over the north Atlantic and north Pacific Oceans (Corbett et al., 1999; Endresen et al., 2003). The North Atlantic shipping lines between North America and Western Europe are amongst the busiest maritime traffic lines, which may have had great influence on the dispersion of G. tigrinus over Europe (Fig. 2B). East Asia has a high potential for the invasion of G. tigrinus as a result of maritime trade with North America and Europe where the G. tigrinus is already a widely distributed species. Within this range, G. tigrinus is most likely to invade coastal areas which in China may be in the provinces of Guangxi, Guangdong, Fujian, Zhejiang, Jiangsu, Shandong, and Liaoning province (Liaodong Peninsula), in Russia the Vladivostok 123

62 Hydrobiologia (2010) 649: Fig. 2 Predicted occurrence of G. tigrinus based on the ecological niche models developed using environmental data for North American sites of G. tigrinus presence. Included in the models are A no dispersal limitations or B addition of the main ocean routes in the world or C limitation based upon 90th percentile (306 km) of reported G. tigrinus dispersal distance in nonnative range or D the potential invasive range of G. tigrinus in Europe. Dispersal distances are measured from the inland location to the nearest coastline. Dot circles indicate occurrence reports of G. tigrinus and crosses indicate some large ports which located in the potential invasive range 123

63 190 Hydrobiologia (2010) 649: region, Korean Peninsula, and southwest Japan (Fig. 2C). The Gulf of Guinea, the coastal areas of Tanzania and Kenya, and the southeast of Madagascar were also predicted as potentially suitable areas as they have medium high environmental matches. In Europe, our model indicates a high invasive potential in the Balkan Peninsula, Apennine Peninsula, and Asia Minor Peninsula, especially the northern coastal areas of the Mediterranean Sea and regions surrounding the Black Sea (Fig. 2D). Potential risk to ports Adult G. tigrinus tolerated the 30 and 45 PSU sodium chloride treatments for h. (Santagata et al., 2008). The salinity of sea water is approximately 36 PSU, yet the salinity of most ports is lower because of input from inland water (Table 3). Gammarus tigrinus has a high invasive risk in Chinese coastal ports and the ports of Buenos Aires and Montevideo (30.42 PSU; Table 3). Shanghai port has the lowest salinity (29.54 PSU) indicating that it may have the highest invasive risk for G. tigrinus and other aquatic organism with a similar niche preference. Ocean ports are not suitable for the survival of G. tigrinus because of the species limited tolerance of salinity (0 25 PSU). Thus, the species is likely to disperse from the port to inland water systems, especially in estuaries. Discussion Ecological niche modeling provides valuable insight into the potential distribution of many nonindigenous species (Peterson, 2003). It can identify areas at risk of invasion, which can focus subsequent management efforts to maximize efficacy. More generally, this study demonstrates the anticipatory capacity of the ENM tools we used. Indeed, even species that have not yet invaded can be evaluated as to their invasive potential, and risks can thus be evaluated proactively (Peterson, 2003). Although certainly not 100% correct in its predictions, the ENM approach offers some information where little or none was available before. This predictive and proactive approach can be used on a broad scale to prioritize use of limited resources in control and prevention of species invasions Table 3 Ports that have a high potential invasive risk for Gammarus tigrinus as determined by our model Countries Ports Invasive risk Salinity (PSU) Argentina Buenos Aires High Australia Adelaide High Brisbane High Hobart High Melbourne High Perth Medium high Sydney High Brazil Rio de Janeiro Medium high Canada Vancouver High China Dalian High Fangchenggang High Fuzhou High Guangzhou High Kaohsiung High Keelung High Lianyungang High Ningbo High Qingdao High Shanghai High Xiamen High Cote d Ivoire Abidjan Medium high Japan Fukuoka Medium high Fukuyama Medium high Hakodate Medium high Hiroshima Medium high Kagoshima Medium high Kanazawa Medium high Kawasaki Medium high Kobe Medium high New Zealand Auckland Medium high Wellington Medium high North Korea Hungnam High Russia Vladivostok High

64 Hydrobiologia (2010) 649: Table 3 continued Countries Ports Invasive risk Salinity (PSU) South Africa Durban Medium high South Korea Busan High Inchon High Uruguay Montevideo High USA Portland High San Francisco High Seattle High Vietnam Haiphong High Salinity data from World Ocean Data 2005, gov (Peterson et al., 2007). GARP is a widely used approach that had succeeded in predicting species distributions accurately in several cases (Peterson & Robins, 2003; Drake & Bossenbroek, 2004; Gaubert et al., 2006). Collectively, these results indicate that GARP models provide valuable insights into potential ranges of nonindigenous species, and this information can be harnessed for the development of management strategies to prevent future invasions (Herborg et al., 2007a). Our GARP model successfully predicted the high potential invasive risk ranges of G. tigrinus, and revealed that most of Europe and East Asia and South America are vulnerable to invasion by G. tigrinus. With the development of the technology, large ships which are used in the ocean shipping accelerated the invasion of aquatic invertebrates transmitted in ballast water. These invasive species reduced the native biological diversity, and as a result of strong environmental tolerance invasive species even became dominant groups in nonnative regions by replacement of native species. Many studies have demonstrated that survival of nonindigenous species depends on the degree of environmental similarity between donor and recipient regions, thereby implicating the importance of physiological tolerance to conditions in the introduced environment (Wonham et al., 2000; Kolar & Lodge, 2002; Rouget & Richardson, 2003; Forsyth et al., 2004). Environmental niche models may yield misleading forecasts of the future ranges of nonindigenous species because successful invasions require a pathway to the potential invasive range. Our results highlight the advantage of combining environmental matching and distribution of larger ports in the marine transportation. Gammarus tigrinus survived at higher temperatures in more ion-rich, polluted waters than the indigenous gammarids, indicating a wider physiological tolerance and thus a higher competitive ability in these waters (Wijnhoven et al., 2003). Domestic water containing more chemical substances may also increase salinity, which may have facilitated the invasion of G. tigrinus of upstream stretches of river systems. Our predictions of potential distribution of G. tigrinus can provide a strong basis for identifying areas where detection efforts would be most effective and beneficial. In Europe, G. tigrinus might have invaded regions of Baltic Sea rapidly because of lower seawater salinity (\10 PSU). Some regions of Western European area where G. tigrinus is found are in relatively low risk from the GARP prediction based on environmental matching. However, as these regions have great maritime trade with native range of G. tigrinus, and received large volume of ballast water from invaded source regions. They have high invasive risk, and G. tigrinus may expand further into regions susceptible to its invasion. Identification of such sites would help in formulating measures to contain its spread. Canals connecting rivers are important pathways for invaders, especially as they connect formerly separated biogeographical regions (Bij de Vaate et al., 2002). In general, the genus of Gammarus is considered widespread in the northern hemisphere but since G. tigrinus was found in the Gulf of Paria and Orinoco Delta in Venezuela (latitude \10 N) (Capelo et al., 2004), it may well cross the equator by ballast water. The Orinoco Delta has rich oil resources and most Venezuelan oil export is to the USA. The invasive G. tigrinus introduced to Orinoco Delta may be a result of this oil trade. Our study suggests that East Asia might be at high risk of invasion by G. tigrinus and other potentially harmful organisms because this area is the focus of intense shipping activity and receives large volume of ballast water. One area in particular is the Yangtze River estuary, which makes it highly susceptible to introductions through intense trading activity. Shanghai is one of the world s financial centers, and the large ports of Shanghai and Ningbo are located on the Yangtze River estuary. Invasive alien freshwater species may quickly disperse to all lower and middle reaches of the Yangtze River after a successful invasion of the ports. 123

65 192 Hydrobiologia (2010) 649: This study also indicates the invasive potential of other freshwater invertebrates with niche preferences similar to G. tigrinus. Because suitable habitats for this species are present on the major continents, particular emphasis should be placed on preventing human-mediated dispersal, especially by ballast water discharges and intentional introductions. The current recommended methods for controlling the introduction of nonindigenous species involve the open ocean exchange of ballast water and the elimination of organisms using chemicals (IMO, 2003; Santagata et al., 2008). This study can assist in detecting specific high invasive risk ports, especially those at high risk of invasions from North America. The further dispersal of G. tigrinus from ports to inland water systems is currently possible, which may result in unforeseeable consequences for ecosystem stability. Efforts should focus on preventing the introduction of exotic aquatic species in these ports. More importantly, high invasive risk ports should have their waters monitored regularly for exotic aquatic organisms; alien species from other countries and continents may survive in these ports either temporarily or permanently. Acknowledgments The article benefited greatly from comments by two anonymous referees. We thank Chengmin Shi, Qingwen Qi, An Zhang and Xi Cheng for their generous helps with GARP and ArcGIS software, and Xinhai Li, Guo Zheng and Yuchi Zheng for critically reading on an earlier version of the manuscript. We thank David M. Lodge (University of Notre Dame, USA) and Leif-Matthias Herborg (BC Ministry of Environment, Canada) for providing useful data. This study was supported by the National Natural Sciences Foundation of China (NSFC / / / ) and National Science Fund for Fostering Talents in Basic Research (Special Subjects in Animal Taxonomy, NSFC-J /J0109). 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68 Zootaxa 2377: 1 93 (2010) Copyright 2010 Magnolia Press Monograph ISSN (print edition) ZOOTAXA ISSN (online edition) ZOOTAXA 2377 The coelotine spiders from three national parks in Northern Vietnam (Araneae, Amaurobiidae) JIE LIU, 1, 3 SHUQIANG LI 1, * & DINH-SAC PHAM 2 1 Institute of Zoology, Chinese Academy of Sciences, Beijing , China. 2 Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hanoi, Vietnam 3 College of Life Sciences, Hubei University, Wuhan , Hubei, China * Corresponding author: lisq@ioz.ac.cn Magnolia Press Auckland, New Zealand Accepted by J. Miller: 15 Feb. 2010; published: 26 Feb. 2010

69 JIE LIU, SHUQIANG LI & DINH-SAC PHAM The coelotine spiders from three national parks in Northern Vietnam (Araneae: Amaurobiidae) (Zootaxa 2377) 93 pp.; 30 cm. 26 February 2010 ISBN (paperback) ISBN (Online edition) FIRST PUBLISHED IN 2010 BY Magnolia Press P.O. Box Auckland 1346 New Zealand Magnolia Press All rights reserved. No part of this publication may be reproduced, stored, transmitted or disseminated, in any form, or by any means, without prior written permission from the publisher, to whom all requests to reproduce copyright material should be directed in writing. This authorization does not extend to any other kind of copying, by any means, in any form, and for any purpose other than private research use. ISSN ISSN (Print edition) (Online edition) 2 Zootaxa Magnolia Press LIU ET AL.

70 Table of contents Abstract Introduction Material and methods Taxonomy Family Amaurobiidae Thorell, Subfamily Coelotinae F.O.P. Cambridge, Genus Coelotes Blackwall, Coelotes acerbus sp. nov Coelotes furvus sp. nov Coelotes perbrevis sp. nov Coelotes polyedricus sp. nov Coelotes songae sp. nov Genus Draconarius Ovtchinnikov, Draconarius clavellatus sp. nov Draconarius cucphuongensis sp. nov Draconarius ellipticus sp. nov Draconarius hanoiensis Wang & Jäger, Draconarius longissimus sp. nov Draconarius magicus sp. nov Draconarius pseudoclavellatus sp. nov Draconarius pseudopumilus sp. nov Draconarius pumilus sp. nov Draconarius rimatus sp. nov Draconarius rotulus sp. nov Draconarius tamdaoensis sp. nov Draconarius transparens sp. nov Draconarius transversus sp. nov Draconarius volubilis sp. nov Genus Notiocoelotes Wang, Xu & Li, Notiocoelotes parvitriangulus sp. nov Notiocoelotes pseudovietnamensis sp. nov Genus Orumcekia Koçak & Kemal, Orumcekia libo (Wang, 2003) Acknowledgments References Abstract Twenty three coelotine species from Northern Vietnam, including twenty one new species, are described and illustrated: Coelotes acerbus sp. nov.; C. furvus sp. nov.; C. perbrevis sp. nov.; C. polyedricus sp. nov.; C. songae sp. nov.; Draconarius clavellatus sp. nov.; D. cucphuongensis sp. nov.; D. ellipticus sp. nov.; D. hanoiensis Wang & Jäger, 2008; D. longissimus sp. nov.; D. magicus sp. nov.; D. pseudoclavellatus sp. nov.; D. pseudopumilus sp. nov.; D. pumilus sp. nov.; D. rimatus sp. nov.; D. rotulus sp. nov.; D. tamdaoensis sp. nov.; D. transparens sp. nov.; D. transversus sp. nov.; D. volubilis sp. nov.; Notiocoelotes pseudovietnamensis sp. nov.; N. parvitriangulus sp. nov.; Orumcekia libo (Wang, 2003). Photos of all twenty three species are provided. All specimens are deposited in the Institute of Zoology, Chinese Academy of Sciences in Beijing (IZCAS). Key words: Taxonomy, diagnosis, morphology, pitfall traps, leaf-litter sieving COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 3

71 Introduction Vietnam stretches more than 1650 kilometers from north to south along Tonkin Bay and the South China Sea, encompassing three major biogeographic zones, four Endemic Bird Areas (EBAs), and a wide variety of unique habitats. Because of its size, location, and the historical interaction of complex topographic, climatic and ecological factors, the country has high levels of species diversity and endemism. The spiders of Vietnam have been studied in recent years (Ono 2002, 2003, 2004a, 2004b; Peng & Li 2003; Tu & Li 2004, 2006; Grismado & Ramírez 2004; Jäger 2003; Jäger & Vedel 2005; Wang & Jäger 2008; Wang, Xu & Li 2008). Lots of new species and newly recorded species were reported. A total 320 spider species and one subspecies were recorded from Vietnam, belonging to 32 families and 159 genera. Among them, 152 species and one subspecies are endemic to Vietnam (Pham et al. 2007). There were only four coelotine species recorded from Vietnam, they are, Draconarius hanoiensis Wang & Jäger, 2008, D. houngsonensis Wang & Jäger, 2008, Notiocoelotes vietnamensis Wang, Xu & Li, 2008, and Orumcekia gemata (Wang, 1994). The first Vietnamese coelotine, C. yoshikoae was reported by Nishikawa (1995), which was later placed as a junior synonym of O. gemata (Wang, 1994) by Wang (2002). The study on Vietnamese coelotine spiders was obviously limited. It is expected that more coelotine spiders exit in Vietnam. An extensive faunal survey of spiders from three national parks (Cuc Phuong National Park, Cat Ba National Park and Tam Dao National Park) in Northern Vietnam over a period of one year using various collection techniques (ground pitfall traps, leaf-litter sieving, canopy fogging and direct search) was carried out by the colleagues of the Chinese Academy of Sciences and Vietnamese Academy of Science and Technology. The collections included 967 adult coelotine spiders representing 23 species, belonging to four genera (Coelotes, Draconarius, Notiocoelotes and Orumcekia), with Draconarius and Coelotes being the most dominant genera, and with D. pumilus sp. nov. (304 adult individuals) and C. polyedricus sp. nov. (40 adult individuals) being the most dominant species of each genus respectively. Among of these 23 species, 21 species are new species, 15 species were collected with only male or female, three species were represented by only one specimen (singletons) (C. songae sp. nov.; D. hanoiensis; O. libo). Of these 23 species, one was unique to Cat Ba National Park, seven to Cuc Phuong National Park, 12 to Tam Dao National Park, three species (D. longissimus sp. nov.; D. transversus sp. nov.; D. volubilis sp. nov.) were distributed in both Cuc Phuong National Park and Tam Dao National Park. All 23 species were described in this paper. Material and methods Study area Total forest cover in Vietnam currently totals 12.3 million ha, of which 10.1 million ha is natural forest and 2.2 million ha is plantations. 36.7% of this forest is protected in National Parks and preserved areas (Wil et al. 2006). The study was carried out in three regions: Cuc Phuong National Park, Tam Dao National Park and Cat Ba National Park in Northern Vietnam. These three parks are approximately 160 km apart (Fig. 87). Cuc Phuong National Park is located from 20º14 20º24 N and 105º29 105º44 E, with an area of 22,200 ha. The park is in Ninh Binh Province, at an elevation of m above the sea level. It is located at the red river delta with a tropical monsoon climate of stable temperatures and humidity gradient. Tam Dao National Park is situated from 21º21 21º42 N and 105º23 105º44 E, in Vinh Phuc Province, with an area of 36,833 ha and an elevation of m. With typical characteristics of high mountain tropical monsoon climate, this park has high humidity and low temperatures. Precipitation comes in the form of mist and rain, and may be accompanied by strong wind (Do 2001). Cat Ba National Park differs from other national parks in Northern Vietnam because it is located on an island in Hai Phong Province. Due to the isolated nature of the island, the diversity and abundance of mam- 4 Zootaxa Magnolia Press LIU ET AL.

72 mals at this park are low compared to other national parks in Vietnam. The park is located from 20º44 20º51 N and 106º58 107º10 E, covering an area of 15,200 ha, at an elevation of m. This national park is affected by maritime climate with weather fluctuation. In addition, typhoons and tropical storms are frequent in the rainy season (Trinh 1985). Study methods Specimens were examined with an Olympus SZ11 stereomicroscope; details were studied with an Olympus BX41 compound microscope. All illustrations were made using an Olympus drawing tube. Male palps and female epigyna were examined and illustrated after being dissected from the spider bodies. Photos were made with an Olympus C7070 wide zoom digital camera (7.1 megapixels) mounted on an Olympus SZX12 Dissecting Microscope. Type specimen photos of the species included in this paper and other related photos can be viewed from Li & Wang (2010). All measurements were obtained using an Olympus SZ11 stereomicroscope and are given in millimeters. Eye diameters were taken at the widest point. Total body length does not include the length of the chelicerae or spinnerets. Leg measurements are given as: Total length (femur, patella + tibia, metatarsus, tarsus). The terminology used in text and figure legends follows Wang (2002) and Wang et al. (2008). Abbreviations used in text and figure legends: ALE = anterior lateral eye; AME = anterior median eye; At = atrium; AME ALE = distance between AME and ALE; AME AME = distance between AMEs; ALE PLE = distance between ALE and PLE; AS = atrial scape; CD = copulatory duct; CDA = conductior dorsal apophysis; CF = cymbial furrow; Co = conductor; EB = embolic base; Em = embolus; ET = Epigynal teeth; FD = fertilization duct; LTA = lateral tibial apophysis; MA = median apophysis; PA = patellar apophysis; PLE = posterior lateral eye; PME = posterior median eye; PME PLE = distance between PME and PLE; PME PME = distance between PMEs; RTA = retrolateral tibial apophysis; S = spermatheca; SH = spermathecal head; ST = subtegulum; T = tegulum; TS = tegulum sclerite. All types of the new species are deposited in the Institute of Zoology, Chinese Academy of Sciences in Beijing (IZCAS). Taxonomy Family Amaurobiidae Thorell, 1870 Subfamily Coelotinae F.O.P. Cambridge, 1893 Genus Coelotes Blackwall, 1841 Diagnosis. Females resemble Eurocoelotes but have laterally situated epigynal teeth, a reduced atrium, and short copulatory ducts. Males also resemble Eurocoelotes in having a conductor dorsal apophysis and a short, rounded median apophysis, but have a broad patellar apophysis (Wang 2002). Distribution: Europe, Middle Asia, East Asia. Coelotes acerbus sp. nov. Figs 1 4, 87 Type material. Holotype male, 2 male paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective acerbus, referring to the sharp distal end of cymbial furrow. Diagnosis. This new species can be distinguished from other Coelotes species by the distinctly large conductor and strongly modified, broad patellar apophysis (Figs 1B, 2A, 3B, 4B). COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 5

73 Description. Male. Total length Holotype total length 7.50, prosoma 4.00 long, 2.75 wide; opisthosoma 3.50 long, 2.35 wide. Eye measurements: AME 0.10; ALE 0.15; PME 0.15; PLE 0.15; AME AME 0.03; AME ALE 0; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (3. 55, 4.00, 3.50, 2.50); II: (3.10, 3.75, 2.90, 1.95); III: (2.80, 3.10, 2.90, 1.55); IV: (3.75, 4.25, 4.00, 1.95). Chelicerae with three promarginal and five retromarginal teeth (Fig. 2B). Patellar apophysis broad, strongly modified (Figs 2A, 4B); RTA with distal end not extending beyond tibia (Figs 2A, 4B); lateral tibial apophysis broad, close to RTA (Figs 2A, 4B); cymbial furrow about half of cymbial length, with distal end sharp, slightly protruding (Figs 2A, 4B); conductor simple, broad, with a triangular-shaped, sharp dorsal apophysis (Figs 1B, 2A, 3B, 4B); median apophysis broad, round (Figs 1B, 2A, 3B, 4B); embolus filiform, originating prolaterally (Figs 1B, 3B). Female. Unknown. Habitat preferences. These specimens were collected by leaf-litter sieving, this species mostly lives in leaf litter. Distribution. Vietnam (Ninh Binh) (Fig. 87). FIGURES 1A B. Coelotes acerbus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, ventral view; Scale bar: 0.2mm. 6 Zootaxa Magnolia Press LIU ET AL.

74 FIGURES 2A B. Coelotes acerbus sp. nov., drawings based on holotype male. A. Left palp, retrolateral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 7

75 FIGURES 3A B. Coelotes acerbus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 8 Zootaxa Magnolia Press LIU ET AL.

76 FIGURES 4A B. Coelotes acerbus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. Coelotes furvus sp. nov. Figs 5 9, 87 Type material. Holotype male, 30 male and 2 female paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective furvus, referring to the black male palps. Diagnosis. This new species can be distinguished from other Coelotes by the uniquely complex conductor which is large and broad, with a folded dorsal apophysis, the cave-shaped tegulum, and the absence of a median apophysis in male (Figs 5B, 6A, 7B, 8B); the anteriorly situated, heart-shaped atrium, absence of epigynal teeth, and large copulatory ducts in female (Figs 6B C, 9B C). Description. Male. Total length Holotype total length 14.25, prosoma 7.00 long, 4.75 wide; opisthosoma 7.25 long, 4.75 wide. Eye measurements: AME 0.20; ALE 0.28; PME 0.25; PLE 0.28; AME AME 0.04; AME ALE 0.05; ALE PLE 0; PME PME 0.10; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (5. 00, 6.00, 4.50, 1.85); II: (4.00, 5.00, 3.75, 2.35); III: COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 9

77 13.10 (3.75, 4.00, 3.35, 2.00); IV: (5.10, 5.75, 5.15, 2.25). Chelicerae with five promarginal and four retromarginal teeth (Fig. 5C). Patellar apophysis long, thin (Figs 5B, 6A, 7B, 8B); RTA with distal end not extending beyond tibia (Figs 5B, 8B); lateral tibial apophysis large, close to RTA (Figs 5B, 8B); cymbial furrow slightly less than half of cymbial length (Figs 5B, 8B); conductor complex, large, conductor dorsal apophysis large, folded (Figs 5B, 6A, 7B, 8B); median apophysis absent (Figs 5B, 6A, 7B, 8B); embolus filiform, originating prolaterally (Figs 6A, 7B). Female. Total length One of paratype total length 14.25, prosoma 6.75 long, 4.65 wide; opisthosoma 8.25 long, 5.75 wide. Eye measurements: AME 0.23; ALE 0.33; PME 0.28; PLE 0.33; AME AME 0.06; AME ALE 0.13; ALE PLE 0; PME PME 0.06; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: [4.65, 5.75, 3.75, 2.30]; II: [4.10, 5.00, 3.15, 2.05]; III: [3.65, 3.75, 3.05, 1.55]; IV: [5.00, 5.75, 4.55, 2.00]. Chelicerae with three promarginal and five retromarginal teeth. Epigynal teeth absent (Figs 6B, 9B); atrium heart-shaped, situated anteriorly (Figs 6B, 9B); copulatory ducts large, folded (Figs 6C, 9C); spermathecal heads absent; spermathecae simple, small, closely set (Figs 6C, 9C). FIGURES 5A C. Coelotes furvus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 10 Zootaxa Magnolia Press LIU ET AL.

78 Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 6A C. Coelotes furvus sp. nov., drawings based on holotype male (A) and female paratype (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 11

79 FIGURES 7A B. Coelotes furvus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 12 Zootaxa Magnolia Press LIU ET AL.

80 FIGURES 8A B. Coelotes furvus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 13

81 FIGURES 9A C. Coelotes furvus sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 1mm. Coelotes perbrevis sp. nov. Figs 10 14, 87 Type material. Holotype male, 1 male and 2 female paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), 6 May 2003, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective perbrevis, referring to the significantly short embolus and conductor. Diagnosis. The new species can be distinguished from other Coelotes species by the short conductor and embolus in male (Figs 11A, 12B), and the oval-shaped, large copulatory ducts in female (Figs 11C, 14C). Description. Male. Total length Holotype total length 10.00, prosoma 5.25 long, 3.75 wide; opisthosoma 4.75 long, 3.25 wide. Eye measurements: AME 0.15; ALE 0.28; PME 0.25; PLE 0.28; AME 14 Zootaxa Magnolia Press LIU ET AL.

82 AME 0.03; AME ALE 0.06; ALE PLE 0; PME PME 0.10; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (4. 75, 5.75, 4.75, 3.00); II: (4.25, 5.25, 4.00, 2.15); III: (3.75, 4.00, 3.60, 2.10); IV: (5.10, 5.50, 5.35, 2.75). Chelicerae with three promarginal and two retromarginal teeth (Fig. 10C). Patellar apophysis broad, large (Figs 10B, 13B); RTA long, slightly shorter than tibial length (Figs 10B, 13B); lateral tibial apophysis wide, close to RTA (Figs 10B, 13B); cymbial furrow less than half of cymbial length (Figs 10B, 13B); conductor extremely short, simple, conductor dorsal apophysis present (Figs 10B, 11A, 12B, 13B); median apophysis broad, round, spoon-shaped (Figs 10B, 11A, 12B, 13B); embolus extremely short and wide, originating prolaterally, arising in a 9 o clock position (Figs 11A, 12B). FIGURES 10A C. Coelotes perbrevis sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. Female. Total length One of paratype total length 10.50, prosoma 5.00 long, 4.15 wide; opisthosoma 5.50 long, 4.00 wide. Eye measurements: AME 0.15; ALE 0.28; PME 0.25; PLE 0.28; AME COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 15

83 AME 0.05; AME ALE 0.08; ALE PLE 0; PME PME 0.10; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: [4.30, 5.25, 4.10, 2.50]; II: [4.25, 5.00, 3.75, 2.40]; III: [3.90, 4.50, 3.80, 2.10]; IV: [5.00, 6.00, 5.35, 2.55]. Chelicerae with three promarginal and two retromarginal teeth. Epigynal teeth small, with sharp end, situated medially and laterally, widely separated (Figs 11B, 14B); atrium small, oval-shaped, situated posteriorly (Figs 11B, 14B); copulatory ducts large and oval-shaped (Figs 11C, 14C); spermathecal heads broad, short, situated anteriorly, widely separated (Figs 11C, 14C); spermathecal stalks long; spermathecal bases simple, small and close to each other (Figs 11C, 14C). Habitat preferences. These specimens were collected in the bark cavities of standing trees. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 11A C. Coelotes perbrevis sp. nov., drawings based on holotype male (A) and female paratype (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. 16 Zootaxa Magnolia Press LIU ET AL.

84 FIGURES 12A B. Coelotes perbrevis sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 17

85 FIGURES 13A B. Coelotes perbrevis sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. 18 Zootaxa Magnolia Press LIU ET AL.

86 FIGURES 14A C. Coelotes perbrevis sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum without skin, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. Coelotes polyedricus sp. nov. Figs 15 19, 87 Type material. Holotype male, 29 male and 10 female paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective polyedricus, referring to the polygonal atrium. Diagnosis. The new species can be distinguished from other Coelotes species by the short and broad retrolateral tibial apophysis, large lateral tibial apophysis which is close to retrolateral tibial apophysis, the absence of cymbial furrow, the unique embolus in male (Figs 15B, 16A, 17B, 18B); by the large and quadrate atrium with two distinct copulatory openings, the unique copulatory ducts in female. (Figs 16B C, 19B C). COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 19

87 Description. Male. Total length Holotype total length 10.15, prosoma 5.25 long, 3.60 wide; opisthosoma 4.90 long, 3.25 wide. Eye measurements: AME 0.13; ALE 0.23; PME 0.23; PLE 0.23; AME AME 0.03; AME ALE 0.04; ALE PLE 0; PME PME 0.08; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (4. 75, 5.75, 4.25, 3.00); II: (4.05, 4.55, 3.30, 2.25); III: (3.75, 4.00, 3.50, 2.05); IV: (4.75, 5.10, 5.00, 2.40). Chelicerae with three promarginal and two retromarginal teeth (Fig. 15C). Patellar apophysis small (Figs 15B, 18B); RTA short, broad, slightly shorter than tibial width (Figs 15B, 18B); lateral tibial apophysis large, close to RTA (Figs 15B, 18B); cymbial furrow absent (Figs 15B, 18B); conductor simple, short, ventral part of conductor narrow, dorsal part wide, conductor dorsal apophysis large (Figs 15B, 16A, 17B, 18B); median apophysis large, spoon-shaped (Figs 16A, 17B); embolus broad proximally and thin distally, originating prolaterally (Figs 16A, 17B). FIGURES 15A C. Coelotes polyedricus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 20 Zootaxa Magnolia Press LIU ET AL.

88 Female. Total length One of paratype total length 10.50, prosoma 4.25 long, 3.00 wide; opisthosoma 6.00 long, 4.10 wide. Eye measurements: AME 0.13; ALE 0.23; PME 0.20; PLE 0.23; AME AME 0.04; AME ALE 0.04; ALE PLE 0; PME PME 0.08; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: [3.55, 4.30, 3.10, 2.05]; II: [3.15, 3.55, 2.60, 1.65]; III: [2.85, 3.00, 2.65, 1.50]; IV: [3.75, 4.25, 3.80, 1.80]. Chelicerae with three promarginal and two retromarginal teeth. Epigynal teeth small, with sharp end, situated anteriorly and laterally, widely separated (Figs 16B,19B); atrium large and quadrate, with two distinct copulatory openings (Figs 16B,19B); copulatory ducts large, extending anteriorly, slightly separated at base and widely separated at distal part (Figs 16C, 19C); spermathecal heads absent (Figs 16C, 19C); spermathecae simple, large and slightly separated at base (Figs 16C, 19C). FIGURES 16A C. Coelotes polyedricus sp. nov., drawings based on holotype male (A) and female paratype (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 21

89 Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 17A B. Coelotes polyedricus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 22 Zootaxa Magnolia Press LIU ET AL.

90 FIGURES 18A B. Coelotes polyedricus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 23

91 FIGURES 19A C. Coelotes polyedricus sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. Coelotes songae sp. nov. Figs 20 23, 87 Type material. Holotype male, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), 3 December 2007, Dinh-Sac PHAM leg. Etymology. The specific epithet is dedicated to Miss SONG Yan-Jing for her kind help to the junior author; noun (name) in apposition. Diagnosis. The new species is similar to Coelotes suthepicus Dankittipakul, Chami-Kranon & Wang, 2005 in having similar patellar apophysis, cymbial furrow, round median apophysis, broad conductor and short embolus (Figs 20B, 21A, 22B, 23B), but can be distinguished from the latter by the following characters: 1, the lateral tibial apophysis line-shaped, close to retrolateral tibial apophysis in this new species, but not in C. suthepicus; 2, the conductor tip reaches the median apophysis proximally in this new species, but far from the median apophysis in C. suthepicus (Figs 20B, 21A, 22B, 23B). Description. Male (measurements of the holotype). Total length prosoma 6.00 long, 3.70 wide; opisthosoma 5.50 long, 3.50 wide. Eye measurements: AME 0.28; ALE 0.25; PME 0.23; PLE 0.25; AME AME 0.08; AME ALE 0.04; ALE PLE 0; PME PME 0.15; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (5.50, 6.75, 5.75, 3.45); II: (5.25, 6.00, 5.10, 3.05); III: (4.90, 5.50, 5.20, 2.55); IV: (6.00, 6.85, 7.30, 3.50). Chelicerae with four promarginal and three 24 Zootaxa Magnolia Press LIU ET AL.

92 retromarginal teeth (Fig. 21B). Patellar apophysis broad and long (Figs 21A, 23B); RTA occupying more than half of the tibial length, distinctly extended distally (Figs 21A, 23B); lateral tibial apophysis broad, lineshaped, close to the RTA (Figs 21A, 23B); cymbial furrow short, less than half of the cymbial length (Figs 21A, 23B); conductor short, broad, concave, dorsal apophysis large (Figs 20B, 21A, 22B, 23B); median apophysis broad, round, spoon-shaped (Figs 20B, 21A, 22B, 23B); embolus short, filiform, prolateral in origin (Figs 20B, 22B). Female. Unknown. Habitat preferences. This specimen was collected by leaf-litter sieving, this species mostly lives in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 20A B. Coelotes songae sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, ventral view. Scale bars: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 25

93 FIGURES 21A B. Coelotes songae sp. nov., drawings based on holotype male. A. Left palp, retrolateral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 26 Zootaxa Magnolia Press LIU ET AL.

94 FIGURES 22A B. Coelotes songae sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 27

95 FIGURES 23A B. Coelotes songae sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. Genus Draconarius Ovtchinnikov, 1999 Diagnosis. This genus resembles Iwogumoa in having an elongated lateral cymbial furrow, a long, slender embolus, and a long, strongly convoluted spermathecae. Females can be distinguished by the posteriorly orig- 28 Zootaxa Magnolia Press LIU ET AL.

96 inated copulatory ducts and the widely separated spermathecae; males by the presence of the conductor dorsal apophysis (Wang 2002). Distribution. Europe, Middle Asia, East Asia. Draconarius clavellatus sp. nov. Figs 24 28, 87 Type material. Holotype male, 120 male and 1 female paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. FIGURES 24A C. Draconarius clavellatus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bar: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 29

97 FIGURES 25A C. Draconarius clavellatus sp. nov., drawings based on holotype male (A) and female paratype (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. Etymology. The specific epithet is taken from the Latin adjective clavellatus, referring to the claviform median apophysis. Diagnosis. The male of new species is similar to Draconarius pseudoclavellatus sp. nov., but can be distinguished from the latter by the following characters: 1, the conductor extends proximally to the median apophysis in this new species (Figs 25A, 26B), the conductor does not extend all the way to the median apophysis in D. pseudoclavellatus sp. nov. (Figs 44B, 46B); 2, the conductor dorsal apophysis thin, claviform in this 30 Zootaxa Magnolia Press LIU ET AL.

98 new species (Figs 24B, 27B), but not in D. pseudoclavellatus sp. nov. (Figs 47B, 45B); 3, the patellar apophysis longer and rounded distally in this new species (Figs 25A, 24B, 26B, 27B), but shorter and acuminated distally in D. pseudoclavellatus sp. nov. (Figs 44B, 45A, 46B, 47B). The males of these two new species can be distinguished from other Draconarius species by the uniquely claviform median apophysis which is extending transversely (Figs 25A, 26B, 44B, 46B). The female of this new species is similar to D. longissimus sp. nov., but can be distinguished from the latter by the presence of spermathecal heads and the different copulatory ducts (Figs 25 B C, 28B C). FIGURES 26A B. Draconarius clavellatus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 31

99 Description. Male. Total length Holotype total length 5.65, prosoma 2.75 long, 2.00 wide; opisthosoma 2.90 long, 2.10 wide. Eye measurements: AME 0.08; ALE 0.15; PME 0.13; PLE 0.13; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (2.50, 3.25, 2.60, 1.70); II: 8.30 (2.20, 2.55, 2.15, 1.40); III: 7.85 (2.25, 2.25, 2.05, 1.30); IV: (2.70, 3.25, 3.00, 1.50). Chelicerae with three promarginal and two retromarginal teeth (Fig. 24C). Patellar apophysis long and thin (Figs 24B, 27B); RTA occupying almost entire tibia length, distinctly extended distally (Figs 24B, 27B); lateral tibial apophysis broad, close to the RTA (Figs 24B, 27B); cymbial furrow long, more than half of the cymbial length (Figs 24B, 27B); conductor simple, with extremely extending posterioly and reaching the median apophysis, conductor dorsal apophysis thin, claviform (Figs 24B, 25A, 26B, 27B); median apophysis long and claviform, with extending transversely (Figs 25A, 26B); embolus long, filiform, retrolateral in origin (Figs 25A, 26B). FIGURES 27A B. Draconarius clavellatus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. 32 Zootaxa Magnolia Press LIU ET AL.

100 Female. Total length prosoma 2.60 long, 2.00 wide; opisthosoma 3.25 long, 2.25 wide. Eye measurements: AME 0.08; ALE 0.15; PME 0.15; PLE 0.15; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, II, III; leg measurements: I: - [-, -, -, -]; II: 7.35 [2.00, 2.50, 1.75, 1.10]; III: 6.70 [1.80, 2.10, 1.80, 1.00]; IV: 9.15 [2.40, 3.00, 2.50, 1.25]. Chelicerae with three promarginal and two retromarginal teeth. Epigynal teeth extremely small, with sharp end, situated posteriorly and laterally, widely separated (Figs 25B, 28B); atrium reduced, line-shaped, slightly close to epigastric furrow (Figs 25B, 28B); copulatory ducts large, extending anteriorly and covering the distal parts of spermathecae (Figs 25C, 28C); spermathecal heads short and small, originating from spermathecae laterally and medianly, widely separated (Figs 25C, 28C); spermathecae large, rounded and close together (Figs 25C, 28C). Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 28A C. Draconarius clavellatus sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 33

101 Draconarius cucphuongensis sp. nov. Figs 29 30, 87 Type material. Holotype female, 2 female paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the type locality, adjective. Diagnosis. The new species is similar to Draconarius huongsonensis Wang & Jäger, 2008 in having the rounded spermathecae, but can be distinguished from the latter by the presence of epigynal teeth, the distinct copulatory ducts and the visible spermathecal heads from dorsal view (Figs 29A B, 30B C). FIGURES 29A C. Draconarius cucphuongensis sp. nov., drawings based on holotype female. A. Epigynum, ventral view; B. Vulva, dorsal view; C. Female cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. Description. Female. Total length Holotype total length 5.00, prosoma 2.25 long, 1.50 wide; opisthosoma 2.75 long, 1.75 wide. Eye measurements: AME 0.05; ALE 0.15; PME 0.13; PLE 0.15; AME AME 0; AME ALE 0.01; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, II, III; leg measurements: I: 5.30 [1.50, 1.75, 1.25, 0.80]; II: 4.75 [1.40, 1.65, 1.00, 0.70]; III: 4.10 [1.20, 1.25, 1.05, 0.60]; IV: 6.10 [1.75, 2.00, 1.65, 0.70]. Chelicerae with three promarginal and two retromarginal teeth (Fig. 29C). Epigynal teeth large, situated posteriorly and laterally, widely separated (Figs 29A, 30B); 34 Zootaxa Magnolia Press LIU ET AL.

102 atrium small, extremely close to epigastric furrow (Figs 29A, 30B); copulatory ducts long, situated between spermathecae (Figs 29B, 30C); spermathecal heads short, situated anteriorly on spermathecae, moderately separated (Figs 29B, 30C); spermathecae large, rounded and slightly separated (Figs 29B, 30C). Male. Unknown. Habitat preferences. These specimens were collected by leaf-litter sieving, this species mostly lives in leaf litter. Distribution. Vietnam (Ninh Binh) (Fig. 87). FIGURES 30A C. Draconarius cucphuongensis sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.25mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 35

103 FIGURES 31A C. Draconarius ellipticus sp. nov., drawings based on holotype female. A. Epigynum, ventral view; B. Vulva, dorsal view; C. Female cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 36 Zootaxa Magnolia Press LIU ET AL.

104 Draconarius ellipticus sp. nov. Figs 31 32, 87 Type material. Holotype female, 1 female paratype, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective ellipticus, referring to the elliptic atrium. Diagnosis. The new species can be distinguished from other Draconarius species by the elliptic atrium and extremely large and unique copulatory ducts (Figs 31A B, 32B C). FIGURES 32A C. Draconarius ellipticus sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 37

105 Description. Female. Total length Holotype total length 9.25, prosoma 4.75 long, 3.20 wide; opisthosoma 4.50 long, 2.75 wide. Eye measurements: AME 0.10; ALE 0.25; PME 0.18; PLE 0.25; AME AME 0.03; AME ALE 0.05; ALE PLE 0.03; PME PME 0.15; PME PLE Clypeus height Leg formula: IV, II, III; leg measurements: I: [3.50, 4.25, 3.00, 1.90]; II: [3.25, 3.50, 2.75, 1.75]; III: [3.00, 3.25, 2.75, 1.50]; IV: [3.50, 4.60, 4.00, 1.90]. Chelicerae with three promarginal and two retromarginal teeth (Fig. 31C). Epigynal teeth small, situated medially and laterally, widely separated (Figs 31A, 32B); atrium elliptic, close to epigastric furrow (Figs 31A, 32B); copulatory ducts large (Figs 31B, 32C); spermathecal heads long and thin, situated on copulatory ducts laterally, widely separated (Figs 31B, 32C); spermathecae long, thin and widely separated (Figs 31B, 32C). Male. Unknown. Habitat preferences. These specimens were collected by leaf-litter sieving, this species mostly lives in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 33A C. Draconarius hanoiensis Wang, 2008, drawings based on holotype female. A. Epigynum, ventral view; B. Vulva, dorsal view; C. Female cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 38 Zootaxa Magnolia Press LIU ET AL.

106 Draconarius hanoiensis Wang & Jäger, 2008 Figs 33 34, 87 D. hanoiensis Wang & Jäger, 2008: 2289, f Material examined. 1 female, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), 23 August 2007, Dinh-Sac PHAM leg. Description. See Wang & Jäger (2008). Habitat preferences. This specimen was collected by leaf-litter sieving, this species mostly lives in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 34A C. Draconarius hanoiensis Wang, 2008, photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 39

107 Draconarius longissimus sp. nov. Figs 35 39, 87 Type material. Holotype male, 110 male and 1 female paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg; 89 male paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective longissimus, referring to the long cymbial furrow and long embolus. Diagnosis. The male of this new species can be distinguished from other Draconarius species by extremely long cymbial furrow and long embolus (Figs 35B, 36A, 37B, 38B). The female of this new species is similar to D. clavellatus sp. nov., but can be distinguished from the latter by the absence of spermathecal heads and the different copulatory ducts (Figs 36C, 39B). FIGURES 35A C. Draconarius longissimus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 40 Zootaxa Magnolia Press LIU ET AL.

108 Description. Male. Total length Holotype total length 6.25, prosoma 3.00 long, 2.15 wide; opisthosoma 3.25 long, 2.30 wide. Eye measurements: AME 0.05; ALE 0.13; PME 0.13; PLE 0.13; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 9.45 (2.65, 3.00, 2.30, 1.50); II: 8.10 (2.35, 2.60, 1.95, 1.20); III: 7.25 (2.05, 2.25, 1.95, 1.00); IV: 9.70 (2.50, 3.25, 2.75, 1.20). Chelicerae with three promarginal and two retromarginal teeth (Fig. 35C). Patellar apophysis small (Figs 35B, 38B); RTA occupying almost entire tibia length (Figs 35B, 38B); lateral tibial apophysis broad, close to the RTA (Figs 35B, 38B); cymbial furrow long, almost as long as the cymbial length (Figs 35B, 38B); conductor simple, extremely broad, with large lamella and small dorsal apophysis (Figs 35B, 36A, 37B, 38B); median apophysis long, spoon-shaped (Figs 35B, 36A, 37B, 38B); embolus extremely long, filiform, retrolateral in origin(figs 36A, 37B). FIGURES 36A C. Draconarius longissimus sp. nov., drawings based on holotype male (A) and female paratype (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. Female. Total length -. prosoma 2.35 long, 1.50 wide; opisthosoma length -, width -. Eye measurements: AME 0.05; ALE 0.13; PME 0.13; PLE 0.13; AME AME 0.01; AME ALE 0; ALE PLE 0; PME PME 0.04; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 5.60 [1.50, 2.00, 1.20, 0.90]; II: 4.60 [1.35, 1.50, 1.00, 0.75]; III: 4.05 [1.25, 1.25, 1.00, 0.55]; IV: 5.80 [1.70, 2.00, 1.50, 0.60]. Chelicerae with three promarginal and two retromarginal teeth. Epigynal teeth extremely small, with sharp end, situated posteriorly and laterally, widely separated (Figs 36B, 39A); atrium reduced, line-shaped (Figs 36B, COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 41

109 39A); copulatory ducts long, extending anteriorly and enlacing the spermathecae (Figs 36C, 39B); spermathecal heads absent (Figs 36C, 39B); spermathecae large, rounded and close together (Figs 36C, 39B). Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Ninh Binh, Vinh Phuc) (Fig. 89). FIGURES 37A B. Draconarius longissimus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 42 Zootaxa Magnolia Press LIU ET AL.

110 FIGURES 38A B. Draconarius longissimus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. FIGURES 39A B. Draconarius longissimus sp. nov., photos based on paratype female. A. Epigynum, ventral view; B. Vulva, dorsal view. Scale bar: 0.5mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 43

111 Draconarius magicus sp. nov. Figs 40 43, 87 Type material. Holotype male, 37 male paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective magicus, referring to the magical and unique male palps. Diagnosis. This new species can be distinguished from other Draconarius species by its uniquely biforked embolus (Figs 40B, 42B). FIGURES 40A B. Draconarius magicus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, ventral view. Scale bar: 0.2mm. Description. Male. Total length Holotype total length 8.50, prosoma 4.25 long, 2.75 wide; opisthosoma 4.25 long, 2.55 wide. Eye measurements: AME 0.13; ALE 0.20; PME 0.18; PLE 0.18; AME AME 0; AME ALE 0.01; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: 44 Zootaxa Magnolia Press LIU ET AL.

112 IV, I, II, III; leg measurements: I: (3.80, 4.75, 3.50, 2.00); II: (3.50, 4.00, 3.20, 2.05); III: (3.20, 3.25, 3.25, 1.75); IV: (4.15, 5.00, 4.60, 2.25). Chelicerae with three promarginal and two retromarginal teeth (Fig. 41B). Patellar apophysis small (Figs 41A, 43B); RTA occupying about half of tibial length (Figs 41A, 43B); lateral tibial apophysis small, widely separated from the RTA (Figs 41A, 43B); cymbial furrow about half of cymbial length (Figs 41A, 43B); conductor simple, with small dorsal apophysis (Figs 40B, 41A, 42B, 43B); median apophysis long, spoon-shaped (Figs 40B, 41A, 42B, 43B); embolus extremely long, broad proximally, thin distally, biforked near the conductor, retrolateral in origin (Figs 40B, 42B). Female. Unknown. Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 41A B. Draconarius magicus sp. nov., drawings based on holotype male. A. Left palp, retrolateral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 45

113 FIGURES 42A B. Draconarius magicus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 46 Zootaxa Magnolia Press LIU ET AL.

114 FIGURES 43A B. Draconarius magicus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. Draconarius pseudoclavellatus sp. nov. Figs 44 47, 87 Type material. Holotype male, 51 male paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet refers to its similarity to D. clavellatus sp. nov. Diagnosis. see the diagnosis under D. clavellatus sp. nov. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 47

115 Description. Male. Total length Holotype total length 7.70, prosoma 4.10 long, 2.75 wide; opisthosoma 3.60 long, 2.30 wide. Eye measurements: AME 0.10; ALE 0.18; PME 0.15; PLE 0.18; AME AME 0.04; AME ALE 0.01; ALE PLE 0; PME PME 0.06; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (3.00, 4.00, 3.10, 2.10); II: (2.75, 3.25, 2.75, 1.55); III: 8.75 (2.60, 2.50, 2.60, 1.05); IV: (3.25, 3.75, 3.50, 1.60). Chelicerae with three promarginal and two retromarginal teeth (Fig. 45B). Patellar apophysis small, with distal end sharp (Figs 45A, 47B); RTA occupying more than half of tibial length, distinctly extended distally (Figs 45A, 47B); lateral tibial apophysis large, close to the RTA (Figs 45A, 47B); cymbial furrow more than half of cymbial length (Figs 45A, 47B); conductor simple, with extremely extending posterioly, conductor dorsal apophysis broad (Figs 44B, 45A, 46B, 47B); median apophysis long and claviform, with extending transversely (Figs 44B, 46B); embolus long, filiform, proximal in origin (Figs 44B, 46B). FIGURES 44A B. Draconarius pseudoclavellatus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, ventral view. Scale bar: 0.2mm. 48 Zootaxa Magnolia Press LIU ET AL.

116 FIGURES 45A B. Draconarius pseudoclavellatus sp. nov., drawings based on holotype male. A. Left palp, retrolateral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. Female. Unknown. Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 49

117 FIGURES 46A B. Draconarius pseudoclavellatus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 50 Zootaxa Magnolia Press LIU ET AL.

118 FIGURES 47A B. Draconarius pseudoclavellatus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. Draconarius pseudopumilus sp. nov. Figs 48 51, 87 Type material. Holotype male, 3 male paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet refers to its similarity to Draconarius pumilus sp. nov. Diagnosis. The new species is similar to Draconarius pumilus sp. nov., but can be distinguished from the latter by the relatively large body size and the absence of navicular conductor dorsal apophysis (Figs 48B, COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 51

119 50A, 51B). This new species is also similar to Draconarius phuhin Dankittipakul, Sonthichai & Wang, 2006 and in having small patellar apophysis, broad conductor, long embolus originating proximally, but can be distinguished from latter by the following characters: 1, the patellar apophysis round distally in this new species, but sharp distally in D. phuhin (Figs 48B, 51B); 2, the cymbial furrow more than half of cymbial length in this new species, but about half of cymbial length in D. phuhin (Figs 48B, 51B). FIGURES 48A B. Draconarius pseudopumilus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 0.2mm. Description. Male. Total length Holotype total length 5.70, prosoma 3.00 long, 1.60 wide; opisthosoma 2.70 long, 1.90 wide. Eye measurements: AME 0.09; ALE 0.18; PME 0.14; PLE 0.14; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.04; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 7.40 (2.15, 2.55, 1.90, 0.80); II: 6.75 (2.00, 2.15, 1.55, 1.05); III: 6.25 (1.80, 2.00, 1.65, 0.80); IV: 8.35 (2.40, 2.55, 2.35, 1.05). Chelicerae with three promarginal and two retromarginal teeth (Fig. 49B). Patellar apophysis small (Figs 48B, 51B); RTA occupying almost entire tibia length, dis- 52 Zootaxa Magnolia Press LIU ET AL.

120 tinctly extended distally; lateral tibial apophysis large, close to the RTA (Figs 48B, 51B); cymbial furrow more than half of cymbial length (Figs 48B, 51B); conductor short and broad, with a large dorsal apophysis (Figs 48B, 49A, 50B, 51B); median apophysis long, spoon-shaped (Figs 48B, 49A, 50B, 51B); embolus long, filiform, proximal in origin (Figs 49A, 50B). FIGURES 49A B. Draconarius pseudopumilus sp. nov., drawings based on holotype male. A. Left palp, ventral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. Female. Unknown. Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Ninh Binh) (Fig. 87). COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 53

121 FIGURES 50A B. Draconarius pseudopumilus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 54 Zootaxa Magnolia Press LIU ET AL.

122 FIGURES 51A B. Draconarius pseudopumilus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 55

123 Draconarius pumilus sp. nov. Figs 52 56, 87 Type material. Holotype male, 269 male and 34 female paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective pumilus, referring to the body size of the new species. Diagnosis. The new species is similar to Draconarius triatus (Wang & Zhu, 1994), but can be distinguished from the by the following characters: 1, D. pumilus sp. nov. is significantly smaller than D. triatus, the former male body is mm long, the latter is mm long; 2, the cymbial furrow more than half of cymbial length in D. pumilus sp. nov., but about half of cymbial length in D. triatus (Figs 52B, 55B); 3, the spermathecal heads thin, small, only seen from a dorsal view in D. pumilus sp. nov., but wide, large, seen from both ventral and dorsal views (Figs 53B C, 56B C). FIGURES 52A C. Draconarius pumilus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bar: 0.2mm. 56 Zootaxa Magnolia Press LIU ET AL.

124 FIGURES 53A C. Draconarius pumilus sp. nov., drawings based on holotype male (A) and female paratype (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. Description. Male. Total length Holotype total length 3.20, prosoma 1.65 long, 1.05 wide; opisthosoma 1.55 long, 1.10 long. Eye measurements: AME 0.04; ALE 0.08; PME 0.08; PLE 0.08; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.03; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 3.65 (1.05, 1.25, 0.80, 0.55); II: 3.15 (1.00, 0.95, 0.70, 0.50); III: 3.35 (0.85, 0.90, 0.70, 0.90); IV: 3.95 (1.20, 1.25, 1.05, 0.45). Chelicerae with five promarginal and five retromarginal teeth (Fig. 52C). Patellar apophysis small (Figs 52B, 55B); RTA occupying almost entire tibia length, distinctly extended distally, with distal end sharp (Figs 52B, 55B); lateral tibial apophysis broad, close to the RTA (Figs 52B, 55B); cymbial furrow more than half of cymbial length (Figs 52B, 55B); conductor short and COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 57

125 broad, with a navicular dorsal apophysis (Figs 53A, 54B); median apophysis long, spoon-shaped (Figs 52B, 53A, 54B, 55B); embolus long, filiform, proximal in origin (Figs 53A, 54B). FIGURES 54A B. Draconarius pumilus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 58 Zootaxa Magnolia Press LIU ET AL.

126 Female. Total length One of paratype total length 2.45, prosoma 1.00 long, 0.80 wide; opisthosoma 1.45 long, 1.00 wide. Eye measurements: AME 0.01; ALE 0.08; PME 0.06; PLE 0.06; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.04; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 2.75 [1.00, 0.90, 0.50, 0.35]; II: 2.10 [0.50, 0.75, 0.50, 0.35]; III: 1.80 [0.60, 0.60, 0.35, 0.25]; IV: 2.55 [0.85, 0.85, 0.50, 0.35]. Chelicerae with five promarginal and five retromarginal teeth. Epigynal teeth small, situated posteriorly and laterally, widely separated (Fig. 53B); atrium small, extremely close to epigastric furrow (Fig. 53B); copulatory ducts long, situated between spermathecae (Figs 53C, 56C); spermathecal heads can be seen from a dorsal view, but not from a ventral view, short and thin, situated anteriorly on spermathecae, widely separated (Figs 53C, 56B C); spermathecae large, rounded and slightly separated (Figs 53C, 56B C). FIGURES 55A B. Draconarius pumilus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 59

127 Habitat preferences. These specimens were collected by both pitfall traps and leaf-litter sieving, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Ninh Binh, Vinh Phuc) (Fig. 87). FIGURES 56A C. Draconarius pumilus sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum without skin, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.25mm. Draconarius rimatus sp. nov. Figs 57 58, 87 Type material. Holotype female, 1 female paratype, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective rimatus, referring to the line-shaped atrium in the new species. 60 Zootaxa Magnolia Press LIU ET AL.

128 Diagnosis. The new species can be distinguished from other Draconarius species by its line-shaped atrium, slightly curved epigynal teeth, thin and long spermathecal heads (Figs 57A B, 58B C). Description. Female. Total length Holotype total length 5.40, prosoma 2.60 long, 1.55 wide; opisthosoma 2.80 long, 2.00 wide. Eye measurements: AME 0.05; ALE 0.15; PME 0.13; PLE 0.15; AME AME 0.03; AME ALE 0.01; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, II, III; leg measurements: I: 7.15 [2.00, 2.50, 1.55, 1.10]; II: 6.30 [1.65, 2.10, 1.50, 1.05]; III: 6.00 [1.75, 1.85, 1.50, 0.90]; IV: 7.95 [2.10, 2.60, 2.25, 1.00]. Chelicerae with three promarginal and two retromarginal teeth (Fig. 57C). Epigynal teeth moderately long, slightly curved, situated anteriorly and laterally, widely separated (Figs 57A, 58B); atrium reduced to a line (Figs 57A, 58B); copulatory broad, extending anteriorly and covering the anterior parts of spermathecae (Figs 57B, 58C); spermathecal heads long and thin, situated anteriorly on spermathecae, extending transversely, slightly separated (Figs 57B, 58C); spermathecae simple, large and slightly separated (Figs 57B, 58C). Male. Unknown. FIGURES 57A C. Draconarius rimatus sp. nov., drawings based on holotype female. A. Epigynum, ventral view; B. Vulva, dorsal view; C. Female cheliceral teeth, ventral view, promargin on the left. Scale bar: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 61

129 Habitat preferences. These specimens were collected by leaf-litter sieving, this species mostly live in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 58A C. Draconarius rimatus sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. Draconarius rotulus sp. nov. Figs 59 62, 87 Type material. Holotype male, 3 male paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective rotulus, referring to the loop-shaped conductor in the new species. Diagnosis. This new species can be distinguished from other Draconarius species by its uniquely huge and loop-shaped conductor (Figs 59B, 61B). 62 Zootaxa Magnolia Press LIU ET AL.

130 Description. Male. Total length Holotype total length 13.00, prosoma 6.75 long, 5.25 wide; opisthosoma 6.25 long, 4.75 wide. Eye measurements: AME 0.25; ALE 0.30; PME 0.25; PLE 0.28; AME AME 0.05; AME ALE 0.08; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (5.00, 5.75, 5.00, 3.25); II: (4.50, 5.85, 4.50, 2.75); III: (4.25, 4.50, 4.00, 2.25); IV: (5.50, 6.00, 5.50, 2.50). Chelicerae with three promarginal and two retromarginal teeth (Fig. 60B). Patellar apophysis broad and large (Figs 60A, 62B); RTA occupying almost entire tibia length (Figs 60A, 62B); lateral tibial apophysis large, close to the RTA (Figs 60A, 62B); cymbial furrow about half of cymbial length (Figs 60A, 62B); conductor extremely long and large, loop-shaped, conductor dorsal apophysis present (Figs 59B, 60A, 61B, 62B); median apophysis broad and short, not spoonshaped (Figs 59B, 60A, 61B, 62B); embolus long, broad proximally, thin and filiform distally, retrolateral in origin (Figs 59B, 61B). Female. Unknown. Habitat preferences. These specimens were collected by leaf-litter sieving, this species mostly live in leaf litter. Distribution. Vietnam (Ninh Binh) (Fig. 87). FIGURES 59A B. Draconarius rotulus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, ventral view. Scale bar: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 63

131 FIGURES 60A B. Draconarius rotulus sp. nov., drawings based on holotype male. A. Left palp, retrolateral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 64 Zootaxa Magnolia Press LIU ET AL.

132 FIGURES 61A B. Draconarius rotulus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 65

133 FIGURES 62A B. Draconarius rotulus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. Draconarius tamdaoensis sp. nov. Figs 63 66, 87 Type material. Holotype male, 15 male paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the type locality; adjective. Diagnosis. This new species can be distinguished from other Draconarius species by its uniquely long conductor, stout median apophysis and small lateral tibial apophysis which is extending tranversely (Figs 63B, 64A, 65B, 66B). Description. Male. Total length Holotype total length 7.90, prosoma 4.25 long, 2.85 wide; opisthosoma 3.65 long, 2.50 wide. Eye measurements: AME 0.11; ALE 0.21; PME 0.18; PLE 0.18; AME AME 0.04; AME ALE 0.03; ALE PLE 0; PME PME 0.10; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (3.75, 4.75, 3.75, 2.25); II: (3.45, 4.15, 3.35, 2.00); III: (3.05, 3.50, 3.40, 1.75); IV: (3.90, 4.75, 4.60, 2.15). Chelicerae with three promarginal and two 66 Zootaxa Magnolia Press LIU ET AL.

134 retromarginal teeth (Fig. 64B). Patellar apophysis short (Figs 64A, 66B); RTA occupying almost entire tibia length, distinctly extended distally (Figs 64A, 66B); lateral tibial apophysis small, extending transversely, widely separated from the RTA (Figs 64A, 66B); cymbial furrow more than half of cymbial length (Figs 64A, 66B); conductor long and large, extending posteriorly and reaching median apophysis, with a strongly curved dorsal apophysis (Figs 63B, 64A, 65B, 66B); median apophysis stout, not spoon-shaped (Figs 63B, 64A, 65B, 66B); embolus long, filiform, proximal in origin (Figs 63B, 65B). Female. Unknown. Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). FIGURES 63A B. Draconarius tamdaoensis sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, ventral view. Scale bar: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 67

135 FIGURES 64A B. Draconarius tamdaoensis sp. nov., drawings based on holotype male. A. Left palp, retrolateral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 68 Zootaxa Magnolia Press LIU ET AL.

136 FIGURES 65A B. Draconarius tamdaoensis sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 69

137 FIGURES 66A B. Draconarius tamdaoensis sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. Draconarius transparens sp. nov. Figs 67 70, 87 Type material. Holotype male, 18 male paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective transparens, referring to the transparent median apophysis in the new species. Diagnosis. The new species is similar to D. clavellatus sp. nov., but can be distinguished from the latter by the transparent median apophysis and almost rectangle-shaped embolus base (Figs 67B, 68A, 69B, 70B). 70 Zootaxa Magnolia Press LIU ET AL.

138 Description. Male. Total length Holotype total length 7.20, prosoma 3.75 long, 2.60 wide; opisthosoma 3.45 long, 2.35 wide. Eye measurements: AME 0.10; ALE 0.16; PME 0.15; PLE 0.15; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (3.00, 3.50, 2.75, 2.00); II: 9.55 (2.50, 3.10, 2.45, 1.50); III: 9.05 (2.50, 2.75, 2.50, 1.30); IV: (3.25, 3.95, 3.50, 1.55). Chelicerae with three promarginal and two retromarginal teeth (Fig. 68B). Patellar apophysis short (Figs 68A, 70B); RTA occupying almost entire tibia length, distinctly extended distally (Figs 68A, 70B); lateral tibial apophysis present, widely separated from the RTA (Figs 68A, 70B); cymbial furrow more than half of cymbial length (Figs 68A, 70B); conductor long and large, extending posteriorly and reaching median apophysis, with a large dorsal apophysis (Figs 67B, 68A, 69B, 70B); median apophysis broad, transparent, not spoon-shaped (Figs 67B, 68A, 69B, 70B); embolus long, filiform, retrolateral in origin (Figs 67B, 69B). Female. Unknown. FIGURES 67A B. Draconarius transparens sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, ventral view. Scale bars: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 71

139 FIGURES 68A B. Draconarius transparens sp. nov., drawings based on holotype male. A. Left palp, retrolateral view; B. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Vinh Phuc) (Fig. 87). 72 Zootaxa Magnolia Press LIU ET AL.

140 FIGURES 69A B. Draconarius transparens sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 73

141 FIGURES 70A B. Draconarius transparens sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. Draconarius transversus sp. nov. Figs 71 75, 87 Type material. Holotype male, 68 male and 3 female paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg; 3 male para- 74 Zootaxa Magnolia Press LIU ET AL.

142 types, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from Latin transversus, referring to the transverse lateral tibial apophysis of the new species; adjective. Diagnosis. The male of this new species can be distinguished from other Draconarius species by two patellar apophysis and stout median apophysis (Figs 71B, 72A, 73B, 74B); the female of this new species is similar to D. hanoiensis, but can be distinguished from the latter by the absence of coiled copulatory ducts (Figs 72C, 75C). FIGURES 71A C. Draconarius transversus sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. Description. Male. Total length Holotype total length 6.05, prosoma 3.25 long, 2.30 wide; opisthosoma 2.80 long, 1.85 wide. Eye measurements: AME 0.08; ALE 0.18; PME 0.15; PLE 0.15; AME AME 0.15; AME ALE 0.01; ALE PLE 0; PME PME 0; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: (3.10, 4.00, 3.00, 2.10); II: (2.75, 3.25, 2.65, 1.75); III: 9.80 (2.55, 3.00, 2.75, 1.50); IV: (3.50, 4.00, 4.25, 1.85). Chelicerae with three promarginal and two retromarginal teeth (Fig. 71C). The male palp with two patellar apophyses, ventral slender and long, dorsal short COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 75

143 and strong (Figs 71B, 74B); the tibia extremely long and slightly curved (Figs 71B, 74B); RTA occupying almost entire tibia length, distinctly extended distally (Figs 71B, 74B); lateral tibial apophysis small, widely separated from the RTA (Figs 71B, 74B); cymbial furrow less than half of cymbial length (Figs 71B, 74B); conductor short and broad, with a sharp distal end and a curved dorsal apophysis (Figs 71B, 72A, 73B, 74B); median apophysis stout, not spoon-shaped (Figs 71B, 72A, 73B, 74B); embolus filiform, prolateral in origin (Figs 72A, 73B). FIGURES 72A C. Draconarius transversus sp. nov., drawings based on holotype male (A) and paratype female (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. 76 Zootaxa Magnolia Press LIU ET AL.

144 FIGURES 73A B. Draconarius transversus sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 77

145 Female. Total length One of paratype total length 5.75, prosoma 2.75 long, 1.90 wide; opisthosoma 3.00 long, 1.80 wide. Eye measurements: AME 0.08; ALE 0.13; PME 0.13; PLE 0.13; AME AME 0.03; AME ALE 0; ALE PLE 0; PME PME 0.08; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 7.10 [2.00, 2.25, 1.75, 1.10]; II: 7.15 [2.00, 2.35, 1.75, 1.05]; III: 6.70 [1.90, 2.15, 1.75, 0.90]; IV: 9.30 [2.50, 3.10, 2.50, 1.20]. Chelicerae with three promarginal and two retromarginal teeth. Epigynum without epigynal teeth (Figs 72B, 75B); atrium small, labiate, close to epigastric furrow (Figs 72B, 75B); copulatory ducts distinct, extending medially and anteriorly between spermathecae, originating from the anterior margin of atrium (Figs 72C, 75C); spermathecae more or less elongated, spermathecal bases widely separated (Figs 72C, 75C); spermathecal heads distinct, anteriorly arising from the copulatory ducts, slightly separated from each other (Figs 72C, 75C). Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Ninh Binh, Vinh Phuc) (Fig. 87). FIGURES 74A B. Draconarius transversus sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. 78 Zootaxa Magnolia Press LIU ET AL.

146 FIGURES 75A C. Draconarius transversus sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. Draconarius volubilis sp. nov. Figs 76 77, 87 Type material. Holotype female, 12 female paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg; 6 female paratypes, VIETNAM: Vinh Phuc Province, Tam Dao National Park (21º31.56 N, 105º33.15 E), March 2007 to March 2008, Dinh-Sac PHAM leg. Etymology. The specific epithet is taken from the Latin adjective volubilis, referring to the voluble copulatory ducts in the new species. Diagnosis. The new species is similar to D. cucphuongensis sp. nov., but can be distinguished from the latter by the relatively small epigynal teeth and the loop-shaped copulatory ducts which are enwinding the spermathecae (Figs 76 A B, 77A B). Description. Female. Total length Holotype total length 6.75, prosoma 3.00 long, 2.05 wide; opisthosoma 3.75 long, 2.75 wide. Eye measurements: AME 0.05; ALE 0.13; PME 0.13; PLE 0.13; AME AME 0.03; AME ALE 0; ALE PLE 0; PME PME 0.05; PME PLE Clypeus height Leg formula: IV, II, III; leg measurements: I: 7.60 [2.25, 2.60, 1.70, 1.05]; II: 6.10 [1.75, 2.00, 1.50, 0.85]; III: 5.70 [1.65, 1.75, 1.50, 0.80]; IV: 7.95 [2.25, 2.75, 2.05, 0.90]. Chelicerae with three promarginal and two retromarginal COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 79

147 teeth (Fig. 76C). Epigynal teeth small, situated posteriorly and laterally, widely separated (Figs 76A, 77B); atrium small, extremely close to epigastric furrow (Figs 76A, 77B); copulatory ducts long, loop-shaped, enlacing the spermathecae, originating posteriorly (Figs 76B, 77C); spermathecal heads short and small, situated anteriorly and laterally on spermathecae, widely separated (Figs 76B, 77C); spermathecae large, rounded and slightly separated (Figs 76B, 77C). Male. Unknown. Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Ninh Binh, Vinh Phuc) (Fig. 87). FIGURES 76A C. Draconarius volubilis sp. nov., drawings based on holotype female. A. Epigynum, ventral view; B. Vulva, dorsal view; C. Female cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 80 Zootaxa Magnolia Press LIU ET AL.

148 FIGURES 77A C. Draconarius volubilis sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: A = 1mm, B C = 0.5mm. Genus Notiocoelotes Wang, Xu & Li, 2008 Diagnosis. Members of Notiocoelotes can be separated from other Coelotinae by the absence of epigynal teeth, the presence of a tongue-shaped atrial scape, and the large copulatory ducts in females, and by the absence of a patellar apophysis, the large, strongly bifurcated lateral tibial apophysis, the absence of a conductor dorsal apophysis, the strongly elongated, coiled conductor, and the reduced or simple median apophysis in males (Wang, Xu & Li 2008). Distribution. East Asia, Southeast Asia. Notiocoelotes parvitriangulus sp. nov. Figs 78 79, 87 Type material. Holotype female, 6 female paratypes, VIETNAM: Hai Phong Province, Cat Ba National Park, Trung Trang Cave (N ', E '), 16 July, 2008, Shuqiang LI, Guo ZHENG & Dinh-Sac PHAM leg. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 81

149 Etymology. The species epithet is derived from the Latin parvi and triangulus, referring to the small and triangular atrium in this new species; adjective. Diagnosis. The new species is similar to N. pseudovietnamensis sp. nov. in having the small and triangular atrium, the large copulatory ducts, the small and anteriorly situated spermathecal heads (Figs 78A B, 79B C), but can be distinguished from the latter by the widely separated and elliptic copulatory ducts, the complex spemathecal stalks which are not entirely covered by copulatory ducts (Figs 78A B, 79B C). Description. Female. Total length Holotype total length 4.75, prosoma 2.25 long, 1.65 wide; opisthosoma 2.50 long, 1.60 wide. Eye measurements: AME 0.05; ALE 0.13; PME 0.11; PLE 0.13; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.08; PME PLE Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 8.60 (2.25, 2.80, 2.20, 1.35); II: 7.35 (1.95, 2.45, 1.75, 1.20); III: 6.60 (1.85, 1.95, 1.80, 1.00); IV: 9.45 (2.45, 3.00, 2.70, 1.30). Chelicerae with three promarginal and two retromarginal teeth (Fig. 78C). Epigynal teeth absent (Figs 78A, 79B); atrium small, triangular, with a tongue-shaped posterior extension (Figs 78A, 79B); copulatory ducts elliptic, covering the most of spermathecae (Figs 784B, 79C); spermathecae large, rounded, widely separated (Figs 78B, 79C); spermathecal heads small, extended anteriorly, with distinct stalks (Figs 78B, 79C). Male. Unknown. FIGURES 78A C. Notiocoelotes parvitriangulus sp. nov., drawings based on holotype female. A. Epigynum, ventral view; B. Vulva, dorsal view; C. Female cheliceral teeth, ventral view, promargin on the left. Scale bar: 0.2mm. 82 Zootaxa Magnolia Press LIU ET AL.

150 Habitat preferences. These specimens were collected in cave, this species may live in the caves or the rock crevices. Distribution. Vietnam (Hai Phong) (Fig. 87). FIGURES 79A D. Notiocoelotes parvitriangulus sp. nov., photos based on holotype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Vulva, dorsal view; D. Female eyes, dorsal view. Scale bars: A = 1mm, B D = 0.2mm. Notiocoelotes pseudovietnamensis sp. nov. Figs 80 84, 87 Type material. Holotype male, 10 male and 6 female paratypes, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), March 2007 to March 2008, Dinh-Sac PHAM leg. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 83

151 Etymology. The specific name refers to its similarity to Notiocoelotes vietnamensis Wang, Xu & Li, Diagnosis. The new species is similar to N. vietnamensis Wang, Xu & Li, 2008 in having the large lateral tibial apophysis, the long cymbial furrow, the long, coiled and semi-circular conductor in male (Figs 80B, 81A, 82B, 83B), in having similar atrium and rounded spermathecae in female (Figs 81B C, 84B D). But can be distinguished from the latter by the absence of an indentation on the distal conductor, the absence of median apophysis, the triangle-shaped embolic base in male (Figs 80B, 81A, 82B, 83B), by the small triangleshaped atrium, and the different shape of copulatory ducts in female (Figs 81B C, 84B D). FIGURES 80A C. Notiocoelotes pseudovietnamensis sp. nov., drawings based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view; C. Male cheliceral teeth, ventral view, promargin on the left. Scale bars: 0.2mm. 84 Zootaxa Magnolia Press LIU ET AL.

152 FIGURES 81A C. Notiocoelotes pseudovietnamensis sp. nov., drawings based on holotype male (A) and female paratype (B C). A. Left palp, ventral view; B. Epigynum, ventral view; C. Vulva, dorsal view. Scale bars: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 85

153 FIGURES 82A B. Notiocoelotes pseudovietnamensis sp. nov., photos based on holotype male. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: A = 1mm, B = 0.2mm. 86 Zootaxa Magnolia Press LIU ET AL.

154 Description. Male. Total length Holotype total length 3.75, prosoma 1.85 long, 1.35 wide; opisthosoma 1.90 long, 1.30 wide. Eye measurements: AME 0.04; ALE 0.11; PME 0.11; PLE 0.11; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.03; PME PLE Eyes pale, AME slightly reduced, the tubercles of eyes not reduced. Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 7.20 (1.90, 2.40, 1.75, 1.15); II: 6.05 (1.70, 1.90, 1.45, 1.00); III: 5.70 (1.55, 1.70, 1.50, 0.95); IV: 7.80 (2.00, 2.45, 2.25, 1.10). Chelicerae with three promarginal and two retromarginal teeth (Fig. 80C). Patellar apophysis absent (Figs 80B, 83B); RTA occupying most of tibial length (Figs 80B, 83B); lateral tibial apophysis large, strongly bifurcated (Figs 80B, 83B); cymbial furrow long, slightly more than half of cymbial length (Figs 80B, 83B); conductor long, extending proximally, coiling, semi-circular, conductor dorsal apophysis absent (Figs 80B, 81A, 82B, 83B); conductor lamella large (Figs 81A, 82B); median apophysis absent (Figs 80B, 81A, 82B, 83B); embolic base large, almost triangular (Figs 81A, 82B); embolus filiform, originating proximally (Figs 81A, 82B). FIGURES 83A B. Notiocoelotes pseudovietnamensis sp. nov., photos based on holotype male. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 0.2mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 87

155 FIGURES 84A D. Notiocoelotes pseudovietnamensis sp. nov., photos based on paratype female. A. Female habitus, dorsal view; B. Epigynum, ventral view; C. Epigynum without skin, ventral view; D. Vulva, dorsal view. Scale bars: A = 1mm, B D = 0.2mm. 88 Zootaxa Magnolia Press LIU ET AL.

156 Female. Total length One of paratype total length 4.00, prosoma 1.75 long, 1.35 wide; opisthosoma 2.25 long, 1.50 wide. Eye measurements: AME 0.04; ALE 0.10; PME 0.10; PLE 0.10; AME AME 0; AME ALE 0; ALE PLE 0; PME PME 0.04; PME PLE Eyes white, AME slightly reduced, the tubercles of eyes slightly reduced. Clypeus height Leg formula: IV, I, II, III; leg measurements: I: 5.30 (1.50, 1.75, 1.20, 0.85); II: 4.50 (1.25, 1.50, 1.05, 0.70); III: 4.40 (1.30, 1.35, 1.10, 0.65); IV: 5.95 (1.55, 2.00, 1.60, 0.80). Chelicerae with three promarginal and two retromarginal teeth. Epigynal teeth absent (Figs 81B, 84B); atrium small, triangular, with a tongue-shaped posterior extension (Figs 81B, 84B); copulatory ducts broad, bilobate, covering the whole spemathecal stalks and heads (Figs 81C, 84C D); spermathecae small, rounded, widely separated, with distal parts covered by copulatory ducts (Figs 81C, 84C D); spermathecal heads small, extended anteriorly, with distinct stalks (Figs 81C, 84C D). Habitat preferences. These specimens were collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. Vietnam (Ninh Binh) (Fig. 87). Genus Orumcekia Koçak & Kemal, 2008 Diagnosis. Females of Orumcekia can be easily distinguished by the absence of epigynal teeth, the presence of a broad, transverse atrial septum, and the posteriorly expanded epigynal posterior margin, males by the presence of two patellar apophyses and a reduced lateral tibial apophysis (Wang 2002). Distribution. China, Vietnam, Thailand. Orumcekia libo (Wang, 2003) Figs 85 86, 87 Coronilla libo Wang, 2003: 99, fig. 99. Orumcekia libo Koçak & Kemal, 2008: 139. Other material: 1 male, VIETNAM: Ninh Binh Province, Cuc Phuong National Park (20º21.44 N, 105º34.21 E), 1 to 30 April 2007, Dinh-Sac PHAM leg. Description. See Wang (2003). Habitat preferences. This specimen was collected by pitfall traps, this species may live on the forest floor or in leaf litter. Distribution. China (Guizhou), Vietnam (Ninh Binh) (Fig. 87). Acknowledgments The manuscript benefited from comments by Xin Ping Wang ( and Jeremy A. Miller (Nationaal Natuurhistorisch Museum Naturalis, Leiden, The Netherlands). This study was supported by the National Natural Sciences Foundation of China (NSFC / / / / / / ), by the National Science Fund for Fostering Talents in Basic Research (Special Subjects in Animal Taxonomy, NSFC-J /J0109), by the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-Z-008/KSCX3-IOZ-0811), by the Ministry of Science and Technology of the People s Republic of China (MOST grant no. 2006FY120100/2006FY110500), and partly also by the Beijing Natural Science Foundation ( ). COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 89

157 FIGURES 85A B. Orumcekia libo (Wang, 2003), photos based on male from Cuc Phuong. A. Male habitus, dorsal view; B. Left palp, ventral view. Scale bars: 1mm. 90 Zootaxa Magnolia Press LIU ET AL.

158 FIGURES 86A B. Orumcekia libo (Wang, 2003), photos based on male from Cuc Phuong. A. Left palp, prolateral view; B. Same, retrolateral view. Scale bar: 1mm. COELOTINE SPIDERS FROM NORTHERN VIETNAM Zootaxa Magnolia Press 91

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