Diversification of Low Dispersal Crustaceans Through Mountain Uplift

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Zoological Journal of the Linnean Society, 2014, 170, 591–633. With 27 ﬁgures

Diversiﬁcation of low dispersal crustaceans through mountain uplift: a case study of Gammarus (Amphipoda: Gammaridae) with descriptions of four novel species

ZHONGE HOU1, JUNBO LI2 and SHUQIANG LI1*

1Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 2School of Life Science, Shanxi Normal University, Linfen 041000, China

Received 22 July 2013; revised 21 November 2013; accepted for publication 23 November 2013

Lineages with low dispersal ability are geographically restricted. We used freshwater Gammarus to test this hypothesis. Sequences of two mitochondrial (cytochrome c oxidase subunit I and 16S) and two nuclear (28S and cytosolic heat-shock protein) genes were obtained for seven species distributed in 28 localities along the Lüliang and Taihang mountains in China. Phylogenetic analyses showed that Gammarus species were grouped into two clades, one from the Lüliang range and the other from the Taihang range. Each clade was further divided into three or four species, showing a congruent pattern with geographical vicariance. Divergence time estimation indicated that the split between the two clades coincided with the uplift of the Taihang Mountains at the boundary of Oligocene/ Miocene. Most speciation events may have been driven by massive uplifting of the Lüliang and Taihang mountains from the late Miocene to early Pliocene. Additionally, four new species are described: Gammarus incoercitus sp. nov., Gammarus benignus sp. nov., Gammarus monticellus sp. nov., and Gammarus pisinnus sp. nov. The new species are compared with related species in this area and a key to these species is provided.

ADDITIONAL KEYWORDS: HSP70 – mtDNA – speciation – taxonomy – time estimation.

INTRODUCTION understanding of the historical processes that forge this correlation continues to develop. Several recent The correlation between phylogeny and geographical studies have demonstrated that geological events, distribution of aquatic organisms with poor dispersal such as mountain uplift, may play an important role ability is widely known (Seidel, Lang & Berg, 2009; in shaping the diversification patterns of animals Shih & Ng, 2011). Extensive planktonic dispersal (Renema et al., 2008; Weir & Price, 2011). is thought to promote gene flow and thus decrease The genus Gammarus Fabricius, 1775, is an ideal genetic differentiation. Therefore, organisms with model to investigate the relationship between the reduced dispersal should have strong structures diversification process and geographical distribution. over small spatial scales (Claramunt et al., 2012). Freshwater Gammarus are characterized by direct Numerous phylogeographical studies have found development of fertilized eggs in a marsupium and that well-supported clades are often distributed in no independent larval stage. Their weak dispersal rather well-defined geographical regions (Fišer, Sket potential means that they are easily influenced by & Trontelj, 2008; Hou et al., 2011). However, our geological barriers, such as mountain ranges, with their distributions often reflecting past geological *Corresponding author. E-mail: [email protected] events (Väinölä et al., 2008). In addition, species

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 591–633 591 592 Z. HOU ET AL. of Gammarus are particularly diversified in cool morphologically and genetically well-defined species running waters such as mountain streams, allowing according to the evolutionary species concept (Mayden, easy sampling for genetic studies. Moreover, most 1997), and we present formal taxonomic descriptions. species of Gammarus are highly endemic to a given landmass. For instance, the species group Gammarus pecos is narrowly endemic to the north-west Gulf MATERIAL AND METHODS of Mexico (Seidel et al., 2009) and the Gammarus SAMPLE COLLECTION fossarum group inhabits Europe (Westram et al., Samples of Gammarus were collected from 28 local- 2011), whereas several species are only found along ities along the Lüliang and Taihang mountain ranges the Lüliang and Taihang mountains in north China. (Fig. 1, Table 1). Freshwater Gammarus lacustris Gammarus shanxiensis Barnard & Dai, 1988 has Sars, 1863, Gammarus decorosus Meng, Hou & Li, been recorded in the southern region of the Taihang 2003, Gammarus komareki Schaferna, 1923, and Mountains (Mts), whereas Gammarus clarus Hou & saline Gammarus aequicauda (Martynov, 1931) were Li, 2010, is endemic to the northern part of the selected as outgroups based on previous phylogenetic Taihang Mts, and Gammarus nekkensis Uchida, 1935, analyses (Hou et al., 2011). exists largely to the far north of the Taihang range. Another four species were newly detected by the present authors during a detailed survey in 2012: DNA SEQUENCE AND PHYLOGENETIC ANALYSES Gammarus incoercitus sp. nov. and Gammarus Genomic DNA was extracted from the heads of speci- benignus sp. nov. were located in the northern and mens using a standard phenol chloroform isoamyl southern parts of the Lüliang Mts; Gammarus protocol (Hillis et al., 1996). Two mitochondrial gene monticellus sp. nov. and Gammarus pisinnus sp. nov. fragments of cytochrome c oxidase subunit I (COI) and were restricted to the south of the Taihang Mts. 16S rRNA with a small part of 12S rRNA, as well as These distinctive geographical patterns suggest that a nuclear gene coding for 28S rRNA were amplified uplifting of the Lüliang and Taihang ranges may have following published protocols (Hou, Fu & Li, 2007). promoted Gammarus speciation events. A fragment of the gene coding for cytosolic heat-shock The orogenic belt of the Taihang Mts runs in a protein (HSP70) was amplified using primers F498 north-to-south direction with an average elevation (5′-GACATGAARCAYTGGCCCTT-3′) and R960 (5′- of 1500–2000 m, dividing the Loess Plateau to the west CGCTTGAAYTCYTGGATGAAGT-3′) (Colson-Proch and the North China Plain to the east. At the early et al., 2010). PCR products were sequenced with the stage of the Miocene (23–16 Mya), the Taihang Mts BigDye terminator sequencing protocols on an ABI were rapidly uplifted to 800 m, followed by a stable 3730 DNA Analyzer. Automated sequence outputs period, then intensely elevated to approximately were imported into SEQUENCHER v. 4.5 (demo 1400 m at the Miocene/Pliocene boundary (6–5 Mya), edition, Gene Codes Corporation) for visual inspection reaching their current elevation at the end of the of the electropherograms. All sequences were aligned Pliocene (Gong, 2010). The Lüliang Mts rose rapidly using MAFFT v. 7.037 (Katoh & Standley, 2013) during the late Miocene (10–6.5 Mya), reaching an and adjusted by eye using MacClade v. 4.0 (Maddison elevation of 1500 m, finally resulting in the connection & Maddison, 2000). For COI and HSP70 sequences, of the Yellow River at 1.6 Mya (Li, 2009). The comple- we checked for the presence of pseudogenes using tion of the Taihang and Lüliang mountains formed the translated amino acid sequences based on Drosophila Shanxi rift between the two ranges. The complex mitochondrial or universal genetic code on MacClade. topography in this region presented a significant geo- The best-fit partitioning schemes and nucleotide graphical divider for the local fauna, and promoted substitution models were selected using Partition- many endemic species (Zhang, Xi & Li, 2006). Surpris- Finder v. 1.1.0 on the Bayesian information criterion ingly, very few investigations have been carried out to (Lanfear et al., 2012). The COI data were partitioned investigate the potential impact of such uplifts on into first, second, and third codon positions with equal- extant biodiversity. frequency Tamura-Nei (TrNef) plus Gamma distrib- In this study, we explored the morphology of uted rate variation among sites (G), transversion Gammarus specimens from the Lüliang and Taihang model (TVM) plus proportion of invariable sites (I), and ranges, and inferred a phylogeny based upon general time reversible with gamma distribution (GTR sequences of two mitochondrial genes and two nuclear + G) substitution models, respectively. The HSP70 genes to unravel the relationships between genetic gene was also partitioned for codon positions, with variation and geographical patterns. We also esti- Felsenstein 1981 (F81) for the first codon, Jukes- mated the divergence time to determine the diversifi- Cantor (JC) for the second codon, and GTR + G for the cation processes associated with the uplifts. In the third codon. The best-fit models for 28S and 16S were course of this study, we detected four undescribed GTR+I+G.

Figure 1. Distribution map of Gammarus species along the Lüliang and the Taihang Mountains (Mts). Numbers and closed circles stand for species and their distributions. The red line represents the separation between the Lüliang Mts clade and the Taihang Mts clade. 1, Gammarus incoercitus sp. nov.;2,Gammarus benignus sp. nov.; 3, Gammarus shanxiensis;4,Gammarus monticellus sp. nov.;5,Gammarus pisinnus sp. nov.;6,Gammarus clarus; 7, Gammarus nekkensis.

We inferred the phylogenetic relationships using constructing a majority consensus tree and estimat- maximum parsimony (MP), maximum likelihood ing the Bayesian posterior probabilities. (ML), and Bayesian inference on both individual and concatenated data sets. MP analysis and bootstrap evaluation were conducted using PAUP* v. 4.0b10 DIVERGENCE TIME ESTIMATION (Swofford, 2002). Alignment gaps were treated as Divergence dates were estimated based on the COI missing data, and phylogenetically uninformative data set. The hypothesis that our data evolved accord- characters were excluded from analysis. Maximum ing to a strict molecular clock was rejected by a likelihood analyses were conducted using RAxML v. likelihood ratio test using PAUP* (lnLnoclock = 7.0.3 (Stamatakis, Hoover & Rougemont, 2008), start- −5424.1, lnLclock = −5456.5, df = 34, P = 0.0011). Con- ing with 1000 rapid bootstrap replications followed by sequently, dating analysis was performed using a a thorough tree search. The MP and ML results did relaxed model. The crustacean mitochondrial COI not indicate conflicting phylogenetic signals for major clock of 0.0115 substitutions per site per million years clades between individual data sets; therefore, we was applied as calibration (Lefébure et al., 2006; Yang, used the concatenated data set in the following analy- Hou & Li, 2013). The analysis was carried out in ses. Bayesian analyses were carried out using BEAST v. 1.7.5 (Drummond & Rambaut, 2007) under MrBayes v. 3.2.1 (Ronquist et al., 2012). The concat- an uncorrelated lognormal relaxed molecular model enated data set was separated into eight partitions and Yule speciation prior. Two independent runs of with each partition following its best-fit substitution 50 000 000 generations were conducted, sampling model. We ran the Monte Carlo Markov chain for every 1000 generations. The resulting files from inde- 8 000 000 generations, sampling every 200 genera- pendent runs were combined using LogCombiner v. tions. The first 20 000 trees were discarded as burn- 1.7.5 and excluded the first 30 000 000 generations in, after checking for stationarity and convergence of as burn-in, resampling every 2000 generations. The the chains in TRACER v. 1.5 (Rambaut & Drummond, maximum clade credibility tree was produced with 2009). We therefore used the last 20 000 trees for the program TreeAnnotator v. 1.7.5 (Drummond &

GenBank accession numbers HOU Z. Altitude Species Locality Voucher (m) Coordinates 28S COI 12–16S HSP70 TAL ET Gammarus incoercitus sp. nov. Pianguan, Xinzhou, China IZCASIA1236 1008 39°24′N, 111°23′E KF824571 KF824601 KF824633 KF824662 G. incoercitus sp. nov. Pianguan, Xinzhou, China IZCASIA1237 1017 39°26′N, 111°24′E KF824572 KF824602 KF824634 KF824663 04TeLnenSceyo London, of Society Linnean The 2014 © G. incoercitus sp. nov. Source of the Fenhe River, Ningwu, China IZCASIA1238 1601 38°49′N, 112°05′E KF824573 KF824603 KF824635 KF824664 . G. incoercitus sp. nov. Source of the Fenhe River, Ningwu, China IZCASIA1240 575 38°48′N, 112°05′E KF824574 KF824604 KF824636 KF824665 G. incoercitus sp. nov. Sanchuan River, Liulin, China IZCASIA1243 805 37°26′N, 110°55′E KF824577 KF824606 KF824639 KF824667 G. incoercitus sp. nov. Huaidao, Ningwu, China IZCASIA1128a 1679 34°40′N, 112°14′E KF824555 KF824588 KF824618 KF824650 Gammarus benignus sp. nov. Wulaofeng Mt., Yongji, China IZCASIA1207 1018 34°48′N, 110°34′E KF824557 − KF824620 − G. benignus sp. nov. Wulaofeng Mt., Yongji, China IZCASIA1207a 1018 34°48′N, 110°34′E KF824556 KF824589 KF824619 KF824651 G. benignus sp. nov. Pangquangou, Fangshan, China IZCASIA1241 1936 37°53′N, 111°26′E KF824576 − KF824638 − G. benignus sp. nov. Pangquangou, Fangshan, China IZCASIA1241a 1936 37°53′N, 111°26′E KF824575 KF824605 KF824637 KF824666 Gammarus shanxiensis Manghe River, Yangcheng, China IZCASIA1212 728 35°16′N, 112°26′E KF824558 KF824590 KF824621 KF824652 G. shanxiensis Yanhe Spring, Yangcheng, China IZCASIA1215 478 35°25′N, 112°31′E KF824559 KF824591 KF824622 KF824653 G. shanxiensis Yangcheng, China IZCASIA519 1370 35°30′N, 112°24′E EF582982 − − − Gammarus monticellus sp. nov. Taiyue Mt., Huozhou, China IZCASIA1253 1829 36°39′N, 112°00′E KF824581 KF824610 − KF824670 G. monticellus sp. nov. Taiyue Mt., Huozhou, China IZCASIA1253a 1829 36°39′N, 112°00′E KF824580 KF824609 KF824642 − G. monticellus sp. nov. Mianshan Mt., Jiexiu, China IZCASIA1247 1558 36°51′N, 112°00′E KF824578 KF824607 KF824640 KF824668 G. monticellus sp. nov. Taotangyu, Huozhou, China IZCASIA1250 1258 36°29′N, 111°51′E KF824579 KF824608 KF824641 KF824669 Gammarus pisinnus sp. nov. Huo Spring, Hongdong, China IZCASIA1254 580 36°18′N, 111°48′E KF824583 KF824612 KF824643 KF824671 olgclJunlo h ina Society Linnean the of Journal Zoological G. pisinnus sp. nov. Huo Spring, Hongdong, China IZCASIA1254a 580 36°18′N, 111°48′E KF824582 KF824611 − − G. pisinnus sp. nov. Nianziguan Spring, Yangquan, China IZCASIA1229 370 37°58′N, 113°52′E KF824564 KF824595 KF824626 KF824656 G. pisinnus sp. nov. Queen Spring, Qinxian, China IZCASIA1227 1034 36°41′N, 112°32′E KF824563 KF824594 KF824625 KF824655 G. pisinnus sp. nov. Chitougou Spring, Yangcheng, China IZCASIA1216 581 35°28′N, 112°32′E KF824560 KF824592 KF824623 KF824654 G. pisinnus sp. nov. Xin’an Spring, Lucheng, China IZCASIA1221 919 36°22′N, 113°23′E KF824562 KF824593 KF824624 − G. pisinnus sp. nov. Xin’an Spring, Lucheng, China IZCASIA1221a 919 36°22′N, 113°23′E KF824561 − − − Gammarus clarus Shentang Spring, Guangling, China IZCASIA1232 960 39°45′N, 114°17′E KF824567 KF824598 KF824629 KF824659 G. clarus Shentang Spring, Guangling, China IZCASIA1233 962 39°45′N, 114°16′E KF824568 KF824599 KF824630 KF824660 G. clarus Source of the Jumahe River, Laiyuan, China IZCASIA315 808 39°18′N, 114°42′E EF582986 EF570340 EF582882 − G. clarus Hutouliang, Yangyuan, China IZCASIA683 1100 40°06′N, 114°06′E EU708621 EU146930 − − Gammarus nekkensis Wutai Mt., Xinzhou, China IZCASIA1230 1401 38°55′N, 113°38′E KF824565 KF824596 KF824627 KF824657 G. nekkensis Wanshui Spring, Wutai, China IZCASIA1231 1664 39°00′N, 113°35′E KF824566 KF824597 KF824628 KF824658 G. nekkensis Wuling Mt., Xinglong, China IZCASIA019 1400 40°36′N, 117°24E KF824587 KF824616 − − G. nekkensis Wuling Mt., Xinglong, China IZCASIA019a 1400 40°36′N, 117°24E KF824585 KF824614 KF824646 KF824674 G. nekkensis Wuling Mt., Xinglong, China IZCASIA019b 1400 40°36′N, 117°24E KF824586 KF824615 KF824647 − G. nekkensis Mengtougou, Beijng, China IZCASIA59 1330 40°00′N, 115°24′E EF582974 EF570331 EF582875 − G. nekkensis Xiaowutai Mt., Yuxian, China IZCASIA337 1200 39°54′N, 115°00′E EU708611 EU146922 − − 2014, , Gammarus lacustris Shentou Spring, Shuozhou, China IZCASIA1235 1056 39°23′N, 112°33′E KF824570 KF824600 KF824631 KF824661 G. lacustris Shentou Spring, Shuozhou, China IZCASIA1235a 1056 39°23′N, 112°33′E KF824569 − KF824632 − G. lacustris Bled, Slovenia SLOCHN001 730 46°21′N, 14°07′E JF965728 JF965915 KF824617 KF824648 170 Gammarus decorosus Shuimogou, Urumqi, China IZCASIA693 867 43°48′N, 87°36′E JF965684 JF965875 KF824645 KF824673

591–633 , Gammarus komareki Kutaisi-Gelati-Tsutskhvati, Georgia IZCASIA1272 253 42°16′N, 42°44′E KF824584 KF824613 KF824644 KF824672 Gammarus aequicauda Patras, Greece SLOCHN065 0 38°08′N, 21°26′E JF965637 JF965832 − KF824649

COI, cytochrome c oxidase subunit I; HSP70, cytosolic heat-shock protein; Mt, Mountain. DIVERSIFICATION OF GAMMARUS SPECIES 595

Rambaut, 2007), summarizing node ages and posterior Taihang Mts clade was composed of four species: probabilities. G. monticellus sp. nov., inhabiting a western branch As mentioned above, the substitution rate of 0.0115 of the Taihang Mts, with an intraspecific divergence is mostly used for the protein-coding gene COI. of 0–10.38% for COI; G. pisinnus sp. nov., widespread In comparison, we performed a regression between in the southern part of the Taihang Mts, with Kimura two-parameter distances for COI vs. the com- an intraspecific divergence of 0.2–12.6% for COI; bined COI and 16S data set (Fig. S1). The slope of G. clarus, located in the northern part of the Taihang this regression was 1.2336, indicating that the com- Mts, mostly found in the upper reaches of rivers bined mitochondrial data set (COI and 16S) evolved or springs, with a low COI intraspecific distance of faster than just COI. We multiplied the COI rate of less than 0.7%; and G. nekkensis, widely distributed 0.0115 by 1.2336 to yield a rate of 0.0142 for the further north, living in the lower reaches in contrast combined mitochondrial data set. The divergence to G. clarus, with a large COI intraspecific distance time estimations for the combined mitochondrial data of up to 18.1%. The interspecific differentiation set were conducted in BEAST with separate substi- amongst the Taihang Mts clade was 11.2–20.3% for tution models for COI and 16S partitions. COI, 1.1–3.7% for 28S, and 1.2–4.1% for HSP70. The COI divergences showed distinctive features, with a higher distance (more than 20%) between the Lüliang MORPHOLOGICAL OBSERVATION Mts and Taihang Mts clades, about 10–20% for Fresh material was preserved in 95% ethanol in the interspecific distances in a clade but less than 10% for field, with selected specimens for morphological obser- intraspecific distances. The same pattern was found vation were transferred to 75% ethanol in the lab. for fragments of 28S and HSP70, with a genetic Body length was recorded by holding the specimen variability of 4–6% between clades, less than 4% for straight and measuring the distance along the dorsal interspecific distance in a clade, and less than 2% for side of the body from the base of the first antenna to intraspecific variability. the base of the telson. Appendages were drawn using Our two approaches with a substitution rate a Leica DM2500 compound microscope equipped with of 0.0115 for COI or 0.0142 for the combined a drawing tube. All types and voucher specimens have mitochondrial data set produced similar divergence been deposited in the Institute of Zoology, Chinese time estimations (Figs 3, S2). For most nodes, the Academy of Sciences (IZCAS), Beijing. 95% posterior credibility intervals of divergence events from two analyses overlapped, but the esti- mates derived from the combined mitochondrial RESULTS analysis were slightly younger for species-level termi- The final alignments contained 41 taxa with 1321 bp nals. The basal split between the Lüliang Mts clade for 28S, 36 taxa with 656 bp for COI, 33 taxa with and Taihang Mts clade was estimated to be 23.3 Mya 982 bp for 16S, and 27 taxa with 434 bp for HSP70. at the boundary of the Oligocene/Miocene. The two The concatenated data set comprised 3393 bp. All new clades started to diversify along their ranges at 16.9 sequences were deposited in GenBank (Table 1). and 19.9 Mya, respectively. The successive diversifi- ML analysis and Bayesian inference yielded a con- cation events occurred from the late Miocene to early gruent topology with high support values for major Pliocene, at 3.4 Mya for G. incoercitus, 5.6 Mya for clades (Fig. 2). The ingroup Gammarus species were G. benignus, 5.7 Mya for G. monticellus, and 6.4 Mya clearly grouped into two clades, corresponding with for G. pisinnus. distinct mountain ranges: a Lüliang Mts clade and a Taihang Mts clade (Figs 1, 2). The Lüliang Mts clade was further divided into three clusters: DISCUSSION G. incoercitus sp. nov., distributed in the northern part of the Lüliang Mts, with an intraspecific uncor- MOUNTAIN UPLIFT PROMOTED rected p-distance of 0–7.2% for COI; G. benignus DIVERSIFICATION EVENTS sp. nov., found in the southern part of the Lüliang The recovered phylogeny strongly supports the Mts, with an intraspecific distance of 9.5% for COI; monophyly of Gammarus species from the Lüliang and G. shanxiensis, restricted to the meeting point and Taihang mountains (Fig. 2). This phylogenetic of Zhongtiao Mt. and the Taihang Mts, with an pattern suggests that Gammarus clades are defined intraspecific divergence of about 1.4% for COI. The by mountain ranges. The timeframe of cladogenetic intraspecific distances for fragments of 28S and events coincides with the uplifts of the Lüliang and HSP70 were less than 2%. The interspecific distances Taihang ranges. Before the Miocene period, this amongst the Lüliang Mts clade were 12.5–19.4% for region was a vast plain (Gong, 2010), and the envi- COI, 1.3–3.2% for 28S, and 1.4–4.4% for HSP70. The ronment was homogeneous. The most recent common

Figure 2. Bayesian tree inferred from the concatenated data set analysis. Names of terminal taxa include voucher numbers for ingroups. The type specimens for nominal species are underlined. Numbers above branches are posterior probabilities; numbers below branches are maximum likelihood bootstrap values. 1, Gammarus incoercitus sp. nov.; 2, Gammarus benignus sp. nov.;3,Gammarus shanxiensis;4,Gammarus monticellus sp. nov.;5,Gammarus pisinnus sp. nov.;6,Gammarus clarus;7,Gammarus nekkensis.

Figure 3. Maximum clade credibility chronogram inferred from a relaxed clock model based on the cytochrome c oxidase subunit I data set. The type specimens for nominal species are underlined. Node bars represent 95% posterior credibility intervals for nodes of interest. 1, Gammarus incoercitus sp. nov.;2,Gammarus benignus sp. nov.;3,Gammarus shanxiensis;4,Gammarus monticellus sp. nov.;5,Gammarus pisinnus sp. nov.;6,Gammarus clarus;7,Gammarus nekkensis. ancestor of the Lüliang and Taihang Mts clades complex topography, higher in the north and lower in colonized freshwater habitats and could exchange the south, closely approximating the genetic diver- freely. Along with the uplift of the Taihang Mts in the gence between G. incoercitus and the cluster of early Miocene, the clades from the Taihang and G. benignus and G. shanxiensis (Fig. 3). The dramatic Lüliang mountains diverged and started to diversify uplift of the Taihang Mts (6–5 Mya) facilitated hetero- (Fig. 3). geneous microhabitats, such as various branches and Most extant species were formed from the late basins, which in turn would generate and sustain Miocene to early Pliocene, probably driven by the diversity in this region. For example, G. monticellus massive uplifts of the Lüliang and Taihang ranges. and G. pisinnus started to radiate across their The rising Lüliang Mts (10–6.5 Mya) resulted in the domains at 5.7 and 6.4 Mya, respectively (Fig. 3).

Between the Lüliang and Taihang mountains, TAXONOMIC IMPLICATIONS the Fen River became fluent in the early Pleisto- cene (1.6 Mya) following the completion of uplifts Molecular analyses indicated that the Lüliang Mts (Li, 2009). The Fen River had little influence on clade consists of three species, G. shanxiensis and Gammarus diversification because the speciation two new species (G. incoercitus and G. benignus). The events pre-dated the formation of contemporary interspecific genetic differentiation for COI reached hydrographical patterns, but it formed a physical 12.5–19.4%, which was sufficient to recognize them as barrier for movement within species across these two independent evolutionary entities compared with the ranges. Gammarus species were adapted to cool value of 4% for Gammarus duebeni (Rock et al., 2007) habitats with relatively high elevations and were not and 8–11% for Gammarus tigrinus (Kelly, MacIsaac & able to cross this river (Ba, Hou & Li, 2011). The Heath, 2006). The interspecific variability for 28S was current patterns of Gammarus species were probably about 4%, which is comparable to results from recent caused by mountain uplifts from the late Miocene research (Hou & Li, 2010). In addition, these species to early Pliocene and maintained by the river’s have allopatric distributions (Fig. 1). Gammarus impediments in the Pleistocene. The distribution shanxiensis is confined to a narrow ecological range of Gammarus extends further westward to Tibet, at the meeting point of two mountains, whereas across several major mountain ranges and river G. incoercitus and G. benignus are distributed in the systems (e.g. Hengduan Mts and Yellow River). northern or southern part of the Lüliang Mts, respec- Further studies should test whether geological tively. The morphology of pereopod 3 of G. incoercitus, ranges are also correlated with distribution patterns which has long, curled setae on the posterior margin, of the genus Gammarus. differs from G. benignus and G. shanxiensis. Gam- marus benignus can be distinguished from its sibling species G. shanxiensis by the short inner ramus of uropod 3. Therefore, molecular, morphological, and ENDEMISM AND LOW DISPERSAL ABILITY ecological data support G. incoercitus and G. benignus The high endemism found in this study confirmed being new species. Gammarus as an extremely rich endemic fauna at The results of this study revealed that four species continental scales. There are about 110 species were nested in the Taihang Mts clade, including two endemic to Europe and western Asia, 16 species ende- known species G. nekkensis and G. clarus, and two mic to North America, and more than 70 species new species G. monticellus and G. pisinnus. These found in East Asia (Väinölä et al., 2008; Hou et al., species demonstrate a interspecific divergence of 2011). Species-level intercontinental distributions are large magnitude (11.2–20.3%) and sharp genetic rare except for G. lacustris, which is distributed discontinuity, suggesting a long period of separate mainly in circum-boreal lakes (Väinölä et al., 2008). evolution. They also inhabit distinctive areas: The present distribution and peculiar endemism may G. monticellus is distributed along the western be attributed to the dispersive capacity of freshwater branch of the Taihang Mts and G. pisinnus is found Gammarus. As it has weak dispersal ability, most of around the margins of the southern basin, whereas the species newly formed at one given location would G. clarus and G. nekkensis occupy the northern parts settle close to their natal location, resulting in local of the Taihang ranges. Moreover, we found that com- endemism. binations of several morphological traits, such as the Studies on other organisms are comparable for armature on the posterior margins of the pereopods endemism. Species with low dispersal ability are and the length of the inner ramus of uropod 3, geographically restricted, and thus they are mostly can help to distinguish between species. Therefore, endemic to local regions. Recent investigations on based on genetic monophyly and morphological diag- freshwater crabs suggest a distinct phylogeographical nosis, we propose G. monticellus and G. pisinnus as pattern in South Africa and East Asia, because fresh- novel species. In addition, the species of G. nekkensis water crabs also lack active dispersal and are highly are deeply divergent, and widely distributed. Other philopatric (Daniels, Gouws & Crandall, 2006; Shih & sources of information, such as mating behaviour and Ng, 2011). In contrast, a study of marine gastropods pheromone preference, need to be explored to verify reveals a negative effect of dispersal on geographical its classification. distribution because their pelagic larvae have greater In conclusion, the phylogeographical patterns potential for dispersal (Lima et al., 2007). Dispersal exhibited by Gammarus provide solid evidence that ability can have powerful impacts on speciation; uplifting of the Lüliang and Taihang mountains therefore, the index of dispersal propensity should be drove the diversification of extant taxa. Furthermore, included in analyses of evolutionary history in further low dispersal ability maintained the high endemism. research. The species clades were geographically defined. The

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 591–633 DIVERSIFICATION OF GAMMARUS SPECIES 599 taxonomy of the genus Gammarus is challenging cute posterodistal corners; inner ramus of uropod 3 because of interspecific morphological similarity. reaching about 0.6 times the length of outer ramus, However, the detection of cryptic species has been outer margin with two groups of spines but few setae. greatly improved with the use of molecular tools and available analytical methods (Seidel et al., 2009; Westram et al., 2011). The findings on these cryptic Description species along with formal morphological investiga- Holotype, male tions will further contribute to biodiversity assess- Body length: 11.3 mm. ment and conservation issues. Head (Fig. 4A): eyes reniform, inferior antennal sinus deep, lateral cephalic lobe nearly straight. Antenna 1 (Fig. 4B, C): peduncular articles 1–3 in SYSTEMATICS length ratio 1.0:0.7:0.4, with setae on distal corners; GAMMARUS INCOERCITUS SP. NOV. (FIGS 4–9) flagellum with 26 articles bearing aesthetascs; acces- Material examined sory flagellum with four articles; both primary and Holotype (IZCAS-I-A1236-1), male, 11.3 mm, a spring accessory flagella with short distal setae. in Fanjia Village (39°24′N, 111°23′E), altitude Antenna 2 (Fig. 4D, E): peduncular articles 3–5 1008 m, Pianguan County, Xinzhou City, Shanxi in length ratio 1.0:3.6:3.8, peduncular articles 4, 5 Province, China, 20.v.2012, collected by J. Li and Z. with clusters of lateral and medial setae; flagellum Chen. Paratypes (from IZCAS-I-A1236-2 to IZCAS-I- with ten articles and one tiny distal article, calceoli A1236-15): eight males and six females, same data as present in articles 1–7. holotype. Upper lip (Fig. 4F): ventral margin rounded, bearing minute setae. Mandible (Fig. 4H, I): left mandible incisor with five Other material teeth; lacinia mobilis with four teeth, spine row with Three males and two females (from IZCAS-I-A1237-1 seven pairs of plumose setae along ventral margin; to IZCAS-I-A1237-5, voucher number 1237), altitude palp articles 1–3 in length ratio 1.0:2.9:1.8; article 2 1017 m, a brook through Fanjia Village (39°26′N, armed with 11 marginal setae; article 3 with three 111°24′E), Pianguan County, Xinzhou City, Shanxi A-setae and two clusters of B-setae, plus 21 D-setae Province, China. 20.v.2012, collected by J. Li and Z. and five E-setae apically; incisor of right mandible with Chen. Eight males and five females (from IZCAS-I- four teeth, lacinia mobilis bifurcate, with small teeth. A1243-1 to IZCAS-I-A1243-13, voucher number 1243), Lower lip (Fig. 4G): inner lobes lacking, outer lobes Sanchuan River (37°26′N, 110°55′E), altitude 805 m, covered with thin setae. Liulin County, Shanxi Province, China, 25.v.2012, Maxilla 1 asymmetrical (Fig. 4J, K), left inner collected by J. Li and Z. Chen. Three males and plate with 14 plumose setae on medial margin; outer three females (from IZCAS-I-A1128a-1 to IZCAS-I- plate with 11 robust serrated apical spines, each A1128a-6, voucher number 1128a), Huaidao Town spines with small teeth; article 2 of palp with seven (38°40′N, 112°14′E), altitude 1679 m, Ningwu County, slender spines and two stiff setae apically; article 2 of Shanxi Province, China, 1.ix.2010, collected by Y. Zong. right palp with six stout spines and one stiff seta and Twelve males and ten females (from IZCAS-I-A1238-1 one slender spine. to IZCAS-I-A1238-22, voucher number 1238), Leiming Maxilla 2 (Fig. 4L): inner plate with 15 plumose Temple (38°49′N, 112°05′E) near the source of the facial setae in an oblique row; inner and outer plates Fenhe River, altitude 1601 m, Ningwu, Shanxi Prov- with long setae apically. ince, China, 23.v.2012, collected by J. Li and Z. Chen. Maxilliped (Fig. 4M): inner plate with three stout Nine males and two females (from IZCAS-I-A1240-1 to apical and one subapical spine, some plumose setae IZCAS-I-A1240-11, voucher number 1240), source of along ventral margin; outer plate bearing a row of the Fenhe River (38°48′N, 112°05′E), altitude 575 m, blade-like spines and five plumose setae apically; palp Ningwu County, Shanxi Province, China, 23.v.2012, article 4 hooked, with a group of setae at hinge of collected by J. Li and Z. Chen. unguis. Gnathopod 1 (Fig. 5A, C): coxal plate bearing two Etymology setae and one seta on anterior and posterior margins, The specific name is from the Latin incoercitus (free), respectively; basis with setae on anterior and posterior referring to freely swimming for this animal. margins; carpus about 1.6 times as long as wide, about 0.7 times as long as propodus, posterior margin Diagnosis bearing short setae; propodus oval, palm with one Merus to carpus of pereopod 3 with long, curled setae medial spine and 14 spines on posterior margin and on posterior margin; epimeral plates 2, 3 with suba- facial surface; dactylus with one seta on outer margin.

Figure 4. Gammarus incoercitus sp. nov., holotype, male. A, head; B, antenna 1; C, aesthetascs of antenna 1; D, antenna 2; E, calceoli of antenna 2; F, upper lip; G, lower lip; H, left mandible; I, incisor of right mandible; J, maxilla 1; K, palp of right maxilla 1; L, maxilla 2; M, maxilliped.

Figure 5. Gammarus incoercitus sp. nov., holotype, male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 6. Gammarus incoercitus 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 7.

Figure 7. Gammarus incoercitus sp. nov., male, A–K; female, L–N. A, epimeral plate 1; B, epimeral plate 2; C, epimeral plate 3; D, urosomites (dorsal view); E, pleopod 1; F, pleopod 2; G, pleopod 3; H, uropod 1; I, uropod 2; J, uropod 3; K, telson; L, uropod 1; M, uropod 2; N, uropod 3.

Figure 8. Gammarus incoercitus sp. nov., female. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 9. Gammarus incoercitus sp. nov., female. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, telson; G, oostegite of gnathopod 2; H, oostegite of pereopod 3; I, oostegite of pereopod 4; J, oostegite of pereopod 5.

Gnathopod 2 (Fig. 5B, D): coxal plate bearing Epimeral plates (Fig. 7A–C): plate 1 ventrally two setae and one seta on anterior and posterior rounded, bearing three setae on anteroventral margin margins, respectively; basis with setae on anterior and three setae on posterior margin; plate 2 with and posterior margins, posterodistal corner with three spines on ventral margin, three setae on pos- short setae; carpus about 1.6 times as long as wide, terior margin, posterodistal corner subacute; plate 3 about 0.8 times as long as propodus, bearing eight with four spines on ventral margin, three setae on clusters of setae along ventral margin, two clusters posterior margin, posterodistal corner acute. of setae on dorsal margin; propodus palm margin Pleopods 1–3 similar (Fig. 7E–G), peduncles with with one medial spine and five spines on lateral one or two retinacula accompanied by one or two posterodistal corner; dactylus with one seta on outer setae; outer ramus slightly shorter than inner ramus, margin. both rami fringed with plumose setae. Pereopod 3 (Fig. 6A, F): coxal plate bearing two Urosomites 1–3 (Fig. 7D): urosomite 1 with setae and one seta on anteroventral and posterior two−two−two spines accompanied by setae on dorsal margins; basis elongate, with setae along anterior margin. Urosomite 2 with three−two−two spines and posterior margins; merus with long, curled accompanied by setae on dorsal margin. Urosomite 3 setae on posterior margin; carpus with groups of with one spine and three spines accompanied by one spines accompanied by long, curled setae on posterior seta on each side. margin; propodus with six groups of spines accompa- Uropods 1–3 (Fig. 7H–J): uropod 1 peduncle with nied by short setae on posterior margin; dactylus with one basofacial spine, with two spines on outer and one plumose seta on posterior margin, and two setae inner margins, two and one spine on outer and inner at hinge of unguis. distal corners, respectively; both rami with two spines Pereopod 4 (Fig. 6B, G): coxal plate excavated, on inner margins and five terminal spines. Uropod 2 bearing two setae on anterior margin and six setae short, peduncle bearing one spine on each margin and on posterior margin; merus with clusters of setae on one distal spine on each corner; inner and outer rami posterior margin; carpus to propodus with groups of both with one spine on inner margins and five termi- spines accompanied by setae on posterior margin; nal spines. Uropod 3 peduncle with one spine on dactylus with one plumose seta on posterior margin, surface and five distal spines; inner ramus about 1.6 and two setae at hinge of unguis. times as long as peduncle, reaching 0.6 times the Pereopod 5 (Fig. 6C, H): coxal plate bearing one seta length of outer ramus, with two spines on inner on anterior and posterior margins, respectively; basis margin, with plumose setae and a few simple setae with five spines on anterior margin, anterodistal along inner and outer margins, bearing two apical corner with two spines, posterior margin with a row spines accompanied by simple setae; article 1 of outer of ten setae; merus to propodus with groups of ramus with three pairs of spines accompanied by spines on anterior margin; dactylus with one plumose simple setae and a few plumose setae on outer seta on posterior margin, and two setae at hinge of margin; terminal article with simple setae, longer unguis. than adjacent spines. Pereopod 6 (Fig. 6D, I): coxal plate bearing one Telson (Fig. 7K): completely cleft, as long as wide, seta on posterior margin; basis elongate, with two each lobe with three clusters of setae and one spine setae and five spines on anterior margin, anterodistal accompanied by setae on surface, with two distal corner with two spines, posterior margin dwindling spines accompanied by setae. distally, with a row of 12 setae; merus to propodus with groups of spines on anterior margin; dactylus Female, paratype (IZCAS-I-A1236-2) with one plumose seta on posterior margin, and two Body length: 9.8 mm. setae at hinge of unguis. Gnathopod 1 (Fig. 8A, C): coxal plate bearing Pereopod 7 (Fig. 6E, J): coxal plate bearing four two setae on anterior margin and one seta on pos- setae on posterior margin; basis with four setae and terior margin; basis with setae on posterior margin; five spines on anterior margin, anterodistal corner propodus oval, palm with six spines on posterior with two spines, posterior margin with a row of 15 margin; dactylus with one seta on outer margin. setae; merus to propodus with groups of spines Gnathopod 2 (Fig. 8B, D): coxal plate bearing two accompanied by a few setae on anterior margin; setae on anterior margin and one seta on posterior dactylus with one plumose seta on posterior margin, margin; basis with short setae on anterior and pos- and two setae at hinge of unguis. terior margins, posterodistal corner with short setae; Coxal gills: coxal gill of gnathopod 2 and gills propodus subrectangular, palm margin with six of pereopods 3–5 a little shorter than bases; gill of spines on posterodistal corner, bearing simple setae pereopod 6 more than half length of basis; gill of along anterior and posterior margins; dactylus with pereopod 7 smallest, less than half of basis. one seta on outer margin.

Pereopods 3, 4 (Fig. 9A, B): with shorter setae on Paratypes (from IZCAS-I-A1207-2 to IZCAS-I-A1207- posterior margin than those of male. 25): 15 males and nine females, same data as holotype. Pereopods 5–7 (Fig. 9C–E): similar to those of male. Uropods 1–3 (Fig. 7L–N): uropods 1 and 2 similar Other material to those of male. Uropod 3 peduncle with one spine Five males and five females (from IZCAS-I-A1241-1 to accompanied by two setae on surface and six distal IZCAS-I-A1241-10, voucher number 1241), Pangquan- spines; inner ramus about 1.3 times as long as pedun- gou Natural Reserve (37°53′N, 111°26′E), altitude cle, reaching 0.6 times the length of outer ramus, with 1936 m, Fangshan County, Shanxi Province, China, one spine and some plumose setae on lateral margin; 24.v.2012, collected by J. Li and Z. Chen. article 1 of outer ramus with two pairs of spines and one single spine accompanied by simple setae and Etymology three plumose setae on outer margin; terminal article The specific name is from the Latin benignus (kind), in longer than adjacent spines. reference to the nice habitat of this species; adjective. Telson (Fig. 9F): cleft, similar to that of male. Oostegite (Fig. 9G–J): oostegite of gnathopod 2 Diagnosis broad, with marginal setae, oostegites of pereopods 3 Eyes oval; pereopod 3 with long, straight setae on and 4 elongate, oostegite of pereopod 5 smallest. posterior margin; epimeral plates 2, 3 with blunt posterodistal corners; uropod 3 slender, inner ramus Habitat reaching about 0.4 times the length of outer ramus. Gammarus incoercitus sp. nov. was found in six localities in northern parts of the Lüliang Mts. Specimens Description were collected near the source of the Fenhe River or Holotype: male upper reaches of brooks with no signs of pollution. Body length: 11.2 mm. Head (Fig. 10A): eyes oval, inferior antennal sinus Remarks deep, lateral cephalic lobe nearly straight. The genus Gammarus is widely distributed through- Antenna 1 (Fig. 10B, C): peduncular articles 1–3 in out the Northern Hemisphere. Up to now, there are length ratio 1.0:0.8:0.4, with distal setae; flagellum about 70 recorded species in China, which are sepa- with 35 articles, most with aesthetascs; accessory rated into north-west and south-east groups driven by flagellum with five articles; both primary and acces- the uplift of the Tibet Plateau (Hou et al., 2007). The sory flagella with short distal setae. species discussed in this paper belong to the south- Antenna 2 (Fig. 10D, E): peduncular articles 3–5 in east group. length ratio 1.0:3.8:3.6, peduncular articles 4, 5 with This new species, G. incoercitus, can be distin- clusters of lateral and medial setae; flagellum with 15 guished from the closely related species G. shanxiensis articles and one tiny distal article, bearing setae along (character states in parentheses) by the following ventral margins; calceoli present in articles 1–8. characters: merus to carpus of pereopod 3 with long, Upper lip (Fig. 10F): ventral margin rounded, curled setae on posterior margin (with short setae bearing minute setae. on posterior margin); epimeral plate 3 with acute Mandible (Fig. 10H, I): left mandible incisor with posterodistal corner (subacute); and inner ramus of five teeth; lacinia mobilis with four teeth, spine row uropod 3 reaching about 0.6 times the length of outer with five pairs of plumose setae along ventral margin; ramus (0.8 times the length of outer ramus). palp articles 1–3 in length ratio 1.0:2.5:1.7; article 2 Gammarus incoercitus differs from Gammarus armed with 15 marginal setae; article 3 with four martensi Hou & Li, 2004 (states in parentheses) by A-setae and two clusters of B-setae, plus 16 D-setae antenna 2 with short setae (densely with long setae); and five E-setae apically; incisor of right mandible epimeral plates 2 and 3 acute (blunt); and urosomites with four teeth, lacinia mobilis bifurcate, with small 1–3 with dorsal spines accompanied by short setae teeth. (with long dorsal setae). Lower lip (Fig. 10G): inner lobes lacking, outer lobes covered with thin setae. Maxilla 1 asymmetrical (Fig. 10J, K), left inner GAMMARUS BENIGNUS SP. NOV. (FIGS 10–15) plate with 13 plumose setae on medial margin; outer Material examined plate with 11 robust serrated apical spines, each Holotype (IZCAS-I-A1207-1), male, 11.2 mm, Wulao- spines with small teeth; article 2 of palp with nine feng Natural Landscape Area (34°48′N, 110°34′E), slender spines and two stiff setae apically; article 2 of altitude 1018 m, Yongji County, Shanxi Province, right palp with five stout spines, one stiff seta and one China, 29.iv.2012, collected by J. Li and Z. Hou. slender spine.

Figure 10. Gammarus benignus sp. nov., holotype, male. A, head; B, antenna 1; C, aesthetascs of antenna 1; D, antenna 2; E, calceoli of antenna 2; F, upper lip; G, lower lip; H, left mandible; I, incisor of right mandible; J, maxilla 1; K, palp of right maxilla 1; L, maxilla 2; M, maxilliped.

Figure 11. Gammarus benignus sp. nov., holotype, male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 12. Gammarus benignus 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 7.

Figure 13. Gammarus benignus sp. nov., male, A–K; female, L–N. A, epimeral plate 1; B, epimeral plate 2; C, epimeral plate 3; D, urosomites (dorsal view); E, pleopod 1; F, pleopod 2; G, pleopod 3; H, uropod 1; I, uropod 2; J, uropod 3; K, telson; L, uropod 1; M, uropod 2; N, uropod 3.

Figure 14. Gammarus benignus sp. nov., female. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 15. Gammarus benignus sp. nov., female. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, telson; G, oostegite of gnathopod 2; H, oostegite of pereopod 3; I, oostegite of pereopod 4; J, oostegite of pereopod 5.

Maxilla 2 (Fig. 10L): inner plate with 14 plumose Pereopod 6 (Fig. 12D, I): coxal plate bearing one facial setae in an oblique row; inner and outer plates seta and three setae on anterior and posterior with long setae apically. margins, respectively; basis elongate, with two clus- Maxilliped (Fig. 10M): inner plate with three stout ters of setae and five spines on anterior margin, apical spines and one subapical spine, some plumose anterodistal corner with two spines, posterior margin setae along ventral margin; outer plate bearing a row dwindling distally, with a row of 17 setae; merus to of blade-like spines and three plumose setae apically; propodus with groups of spines on anterior margin, palp article 4 hooked, with a group of setae at hinge propodus with three groups of setae on posterior of unguis. margin; dactylus with one plumose seta on posterior Gnathopod 1 (Fig. 11A, C): coxal plate bearing three margin, and two setae at hinge of unguis. and five setae on anterior and posterior margins, Pereopod 7 (Fig. 12E, J): coxal plate bearing five respectively; basis with setae on anterior and pos- setae on posterior margin; basis with two clusters of terior margins; carpus about 1.3 times as long as setae and four spines on anterior margin, anterodistal wide, about 0.7 times as long as propodus, posterior corner with two spines, posterior margin with a margin bearing short setae; propodus oval, palm with row of 18 setae, inner surface with one spine accom- one medial spine and 18 spines on posterior margin panied by one seta; merus to propodus with groups and facial surface, respectively; dactylus with one of spines accompanied by few setae on anterior seta on outer margin. margin, propodus with two clusters of setae on pos- Gnathopod 2 (Fig. 11B, D): coxal plate bearing four terior margin; dactylus with one plumose seta on setae on anterior margin and four setae on posterior posterior margin, and two setae at hinge of unguis. margin; basis with setae on anterior and posterior Coxal gills: coxal gill of gnathopod 2 and gills margins, posterodistal corner with short setae; carpus of pereopods 3–5 a little shorter than bases; gill of about 1.7 times as long as wide, about 0.8 times as long pereopod 5 more than half length of basis; gill of as propodus, with parallel margins, bearing eight pereopod 7 smallest, less than half of basis. clusters of setae along ventral margin, two clusters of Epimeral plates (Fig. 13A–C): plate 1 ventrally setae on dorsal margin; propodus palm ovate, palm rounded, bearing four long setae and four short setae margin with one medial spine and five spines on on anteroventral margin, with four setae on posterior posterodistal corner; dactylus with one seta on outer margin; plate 2 with two spines on ventral margin and margin. three setae on posterior margin, posterodistal corner Pereopod 3 (Fig. 12A, F): coxal plate bearing four blunt; plate 3 with four spines on ventral margin and setae and three setae on anteroventral and posterior six setae on posterior margin, posterodistal corner margins, respectively; basis elongate, with short blunt. setae along anterior and posterior margins; merus to Pleopods 1–3 similar (Fig. 13E–G), peduncles with carpus with long, straight setae on posterior margin; one or two retinacula accompanied by one setae; inner carpus with groups of spines on posterior margin; and outer rami fringed with plumose setae. propodus with five groups of spines accompanied by Urosomites 1–3 (Fig. 13D): urosomite 1 with one− short setae on posterior margin; dactylus with one two−one spine accompanied by setae on dorsal margin. plumose seta on posterior margin and two setae at Urosomite 2 with two−one−one−two spines accompa- hinge of unguis. nied by setae on dorsal margin. Urosomite 3 with two Pereopod 4 (Fig. 12B, G): coxal plate excavated, spines accompanied by one seta on each side. bearing three setae on anterior margin and seven setae Uropods 1–3 (Fig. 13H–J): uropod 1 peduncle with on posterior margin; basis with one spine accompanied one basofacial spine, three spines on outer margin and by short setae on anterodistal corner; merus with one spine on inner margin, with two and one spines on clusters of setae on posterior margin and one spine on outer and inner distal corners, respectively; outer anterior margin, anterodistal corner with one spine ramus with two spines on outer margin and one spine accompanied by some setae; carpus and propodus with on inner margin; inner ramus with one spine on inner groups of spines accompanied by short setae on pos- margin; both rami with five terminal spines. Uropod 2 terior margin; dactylus with one plumose seta on short, peduncle bearing two spines on outer margin, anterior margin and two setae at hinge of unguis. with one distal spine on each corner; outer ramus with Pereopod 5 (Fig. 12C, H): coxal plate bearing four one spine on outer margin and one spine on inner setae on posterior margin; basis with two clusters of margin; inner ramus with two spines on inner margin. setae and six spines on anterior margin, anterodistal Uropod 3 slender, peduncle with one spine accompa- corner with two spines, posterior margin with a row nied by two setae on surface and five distal spines; of 12 setae; merus to propodus with groups of spines inner ramus about 1.2 times as long as peduncle, on anterior margin; dactylus with one plumose seta reaching 0.4 times the length of outer ramus, with on posterior margin and two setae at hinge of unguis. one spine accompanied by plumose setae on inner

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 591–633 DIVERSIFICATION OF GAMMARUS SPECIES 615 margin, bearing three apical spines accompanied by pereopod 3 with long, straight setae on posterior simple setae; article 1 of outer ramus with two pairs of margin (with short setae on posterior margin); spines and one single spine on outer margin, both epimeral plates 2, 3 posterodistal corners blunt (suba- margins with plumose setae; terminal article slightly cute); and uropod 3 slender, inner ramus reaching shorter than adjacent spines. about 0.4 times the length of outer ramus (0.8 times Telson (Fig. 13K): deeply cleft, as long as wide, each the length of outer ramus). lobe with one spine accompanied by three setae and Gammarus benignus sp. nov. differs from G. clusters of setae on surface, bearing two distal spines incoercitus (character states in parentheses) by eyes accompanied by five setae. oval (reniform); pereopod 3 with long, straight setae on posterior margin (curled setae); and inner ramus Female: paratype (IZCAS-I-A1207-2) reaching about 0.4 times the length of outer ramus Body length: 10.5 mm. (0.6 times the length of outer ramus). Gnathopod 1 (Fig. 14A, C): coxal plate bearing four setae on anterior margin and four setae on posterior GAMMARUS MONTICELLUS SP. NOV. (FIGS 16–21) margin; basis with setae on anterior and posterior margins; propodus oval, palm with seven spines on Material examined posterior margin; dactylus with one seta on outer Holotype (IZCAS-I-A1253-1), male, 12.1 mm, Taiyue ′ ′ margin. Mt National Forest Park (36°39 N, 112°00 E), alti- Gnathopod 2 (Fig. 14B, D): coxal plate bearing tude 1829 m, Linfen City, Shanxi Province, China, five setae and four setae on anterior and posterior 30.v.2012, collected by J. Li and Z. Chen. Paratypes margins, respectively; basis with setae on anterior (from IZCAS-I-A1253-2 to IZCAS-I-A1253-20), ten and posterior margins, posterodistal corner with males and nine females, same data as holotype. short setae; propodus subrectangular, palm margin with four spines on posterodistal corner, bearing Other material simple setae along anterior and posterior margins; Eight males and five females (from IZCAS-I-A1250- dactylus with one seta on outer margin. 1 to IZCAS-I-A1250-13, voucher number 1250), ′ ′ Pereopods 3, 4 (Fig. 15A, B): with shorter setae on Taotangyu (36°29 N, 111°51 E), altitude 1258 m, posterior margin than those of male. Huozhou City, Shanxi Province, China, 28.v.2012, col- Pereopods 5–7 (Fig. 15C–E): similar to those of male. lected by J. Li and Z. Chen. Fifteen males and six Uropods 1–3 (Fig. 13L–N): uropods 1 and 2 similar females (from IZCAS-I-A1250-1 to IZCAS-I-A1250-21, ′ to those of male. Uropod 3 peduncle with one spine voucher number 1247), Mianshan Mt (36°51 N, ′ accompanied by setae on surface and four distal 112°00 E), altitude 1558 m, Jiexiu City, Shanxi Prov- spines; inner ramus as long as peduncle, reaching 0.4 ince, China, 26.v.2012, collected by J. Li and Z. Chen. times the length of outer ramus, with one spine and plumose setae on inner margin; article 1 of outer Etymology ramus with two pairs of spines and one single spine The specific name is from the Latin monticellus accompanied by simple and plumose setae on outer (mountain), in reference to the habitat of this species; margin, inner margin with one spine and plumose noun. setae; terminal article longer than adjacent spines. Telson (Fig. 15F): cleft, similar to that of male. Diagnosis Oostegite (Fig. 15G–J): oostegite of gnathopod 2 Merus to carpus of pereopod 3 with long, curled setae broad, with marginal setae, oostegites of pereopods 3 on posterior margin; epimeral plates 2, 3 with blunt and 4 elongate, oostegite of pereopod 5 smallest. posterodistal corners; inner ramus of uropod 3 reaching about 0.6 times length of outer ramus, both Habitat margins densely set with plumose setae. This species was found in two sites in southern parts of the Lüliang Mts. Samples were collected along Description brooks flowing from the peak of the mountain, under Holotype: male rotten leaves. However, they are absent near the Body length: 12.1 mm. entrance of the scenic area, probably because of the Head (Fig. 16A): eyes reniform, inferior antennal vast numbers of tourists. sinus deep, lateral cephalic lobe nearly straight. Antenna 1 (Fig. 16B, C): peduncular articles 1–3 in Remarks length ratio 1.0:0.8:0.4, with distal setae; flagellum Gammarus benignus sp. nov. can be distinguished with 35 articles, most with aesthetascs; accessory from its sibling species G. shanxiensis (character flagellum with four articles; both primary and acces- states in parentheses) by the following characters: sory flagella with short distal setae.

Figure 16. Gammarus monticellus sp. nov., holotype, male. A, head; B, antenna 1; C, aesthetascs of antenna 1; D, antenna 2; E, calceoli of antenna 2; F, upper lip; G, lower lip; H, left mandible; I, incisor of right mandible; J, maxilla 1; K, palp of right maxilla 1; L, maxilla 2; M, maxilliped.

Figure 17. Gammarus monticellus sp. nov., holotype, male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 18. Gammarus monticellus 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 7.

Figure 19. Gammarus monticellus sp. nov., male, A–K; female, L–N. A, epimeral plate 1; B, epimeral plate 2; C, epimeral plate 3; D, urosomites (dorsal view); E, pleopod 1; F, pleopod 2; G, pleopod 3; H, uropod 1; I, uropod 2; J, uropod 3; K, telson; L, uropod 1; M, uropod 2; N, uropod 3.

Figure 20. Gammarus monticellus sp. nov., female. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 21. Gammarus monticellus sp. nov., female. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, telson; G, oostegite of gnathopod 2; H, oostegite of pereopod 3; I, oostegite of pereopod 4; J, oostegite of pereopod 5.

Antenna 2 (Fig. 16D, E): peduncular articles 3–5 in posterior margin; carpus to propodus with groups of length ratio 1.0:2.8:2.6, peduncular articles 4, 5 with spines accompanied by long, curled setae on posterior clusters of lateral and medial setae; flagellum with 13 margin; dactylus with one plumose seta on posterior articles and one tiny distal article; calceoli present in margin and two setae at hinge of unguis. articles 1–8. Pereopod 4 (Fig. 18B, G): coxal plate excavated, Upper lip (Fig. 16F): ventral margin rounded, bearing three setae on anterior margin and four setae bearing minute setae. on posterior margin; basis with long setae on pos- Mandible (Fig. 16H, I): left mandible incisor with terior margin; merus with clusters of straight setae five teeth; lacinia mobilis with four teeth, spine row on posterior margin; carpus to propodus with groups with six pairs of plumose setae along ventral margin; of spines accompanied by setae on posterior margin; palp articles 1–3 in length ratio 1.0:2.7:1.9; article 2 dactylus with one plumose seta on posterior margin armed with nine marginal setae; article 3 with four and two setae at hinge of unguis. A-setae and two clusters of B-setae, plus 18 D-setae Pereopod 5 (Fig. 18C, H): coxal plate bearing one and five E-setae apically; incisor of right mandible with seta on anterior margin and four setae on posterior four teeth, lacinia mobilis bifurcate, with small teeth. margin; basis with one seta and five spines on ante- Lower lip (Fig. 16G): inner lobes lacking, outer rior margin, anterodistal corner with one spine, lobes covered with thin setae. posterior margin with a row of 11 setae; merus to Maxilla 1 asymmetrical (Fig. 16J, K), left inner propodus with groups of spines on anterior margin; plate with 16 plumose setae on medial margin; outer dactylus with one plumose seta on posterior margin plate with 11 robust, serrated apical spines, each and two setae at hinge of unguis. spines with small teeth; article 2 of palp with nine Pereopod 6 (Fig. 18D, I): coxal plate bearing three slender spines apically; article 2 of right palp with setae on posterior margin; basis elongate, with three five stout spines, one stiff seta and one slender spine. seta and four spines on anterior margin, anterodistal Maxilla 2 (Fig. 16L): inner plate with 17 plumose corner with two spines, posterior margin dwindling setae in an oblique row; inner and outer plates with distally, with a row of 12 setae; merus to propodus long setae apically. with groups of spines on anterior margin; dactylus Maxilliped (Fig. 16M): inner plate with three stout with one plumose seta on posterior margin and two apical spines and one subapical spine, some plumose setae at hinge of unguis. setae along ventral margin; outer plate bearing a row Pereopod 7 (Fig. 18E, J): coxal plate bearing four of blade-like spines and four plumose setae apically; setae on posterior margin; basis with four setae and palp article 4 hooked, with a group of setae at hinge five spines on anterior margin, anterodistal corner of unguis. with one spine, posterior margin with a row of 13 Gnathopod 1 (Fig. 17A, C): coxal plate bearing setae, inner surface with two spines; merus to three setae and one seta on anterior and posterior propodus with groups of spines on anterior margin; margins, respectively; basis with setae on anterior dactylus with one plumose seta on posterior margin and posterior margins; carpus about 1.4 times as long and two setae at hinge of unguis. as wide, about 0.6 times as long as propodus, pos- Coxal gills: coxal gill of gnathopod 2 and gills terior margin bearing short setae; propodus oval, of pereopods 3–5 a little shorter than bases; gill of palm with one medial spine and 13 spines on pos- pereopod 6 more than half length of basis; gill of terior margin and facial surface; dactylus with one pereopod 7 smallest, less than half of basis. seta on outer margin. Epimeral plates (Fig. 19A–C): plate 1 ventrally Gnathopod 2 (Fig. 17B, D): coxal plate bearing rounded, bearing two setae on anteroventral margin, three setae and one seta on anterior and posterior three setae on posterior margin; plate 2 with one seta margins, respectively; basis with long setae on ante- and three spines on ventral margin, four setae on rior and posterior margins, posterodistal corner with posterior margin, posterodistal corner blunt; plate 3 short setae; carpus about 1.7 times as long as wide, with two spines on ventral margin, two setae on about 0.9 times as long as propodus, with parallel posterior margin, posterodistal corner blunt. margins, bearing nine clusters of setae along ventral Pleopods 1–3 similar (Fig. 19E–G), peduncles with margin and two clusters of setae on dorsal margin; one or two retinacula accompanied by one or two propodus palm ovate, palm margin with one medial setae; outer ramus slightly shorter than inner ramus, spine and five spines on lateral posterodistal corner; both rami fringed with plumose setae. dactylus with one seta on outer margin. Urosomites 1–3 (Fig. 19D): Urosomite 1 with two− Pereopod 3 (Fig. 18A, F): coxal plate bearing three one−one−two spines accompanied by setae on dorsal setae and one seta on anteroventral and posterior margin. Urosomite 2 with one−one−one−two spines margins, respectively; basis with long setae along accompanied by setae on dorsal margin. Urosomite 3 posterior margin; merus with long, curled setae on with two spines accompanied by one seta on each side.

Uropods 1–3 (Fig. 19H–J): uropod 1 peduncle with Oostegite (Fig. 21G–J): oostegite of gnathopod 2 one basofacial spine, with three spines on outer broad, with marginal setae, oostegites of pereopods 3 margin, one spine on inner margin, two and one spines and 4 elongate, oostegite of pereopod 5 smallest. on outer and inner distal corners, respectively; inner and outer rami both with one spine on inner margins Habitat and five terminal spines. Uropod 2 short, peduncle The species is currently known from three localities in bearing two spines on outer margin, one spine on each Taiyue Mt., a southern branch of the Taihang Mts; corner; outer ramus with one spine on outer margin; where it occurs at the banks of small streams. inner ramus with one spine on inner margin. Uropod 3 peduncle with one spine accompanied by one seta on surface and four distal spines; inner ramus about 1.6 Remarks times as long as peduncle, reaching 0.6 times the Gammarus monticellus sp. nov. can be distinguished length of outer ramus, with one spine on inner margin from G. shanxiensis (character states in parentheses) and two apical spines accompanied by simple setae; by the following characters: merus to carpus of article 1 of outer ramus with two pairs of spines and pereopod 3 with long, curled setae on posterior margin one single spine on outer margin; terminal article (with short setae on posterior margin); epimeral plates longer than adjacent spines, both margins of inner and 2, 3 with blunt posterodistal corners (subacute); and outer ramus densely set with plumose setae. inner ramus of uropod 3 reaching about 0.6 times the Telson (Fig. 19K): deeply cleft, as long as wide, each length of outer ramus (0.8 times length of outer lobe with clusters of setae and one spine accompanied ramus). by two setae on surface, bearing two or three apical This species is similar to G. clarus in calceoli present spines accompanied by setae. in antenna 2 and both margins of uropod 3 densely set with plumose setae. It differs from G. clarus (character Female, paratype (IZCAS-I-A1253-2) states in parentheses) by pereopod 3 with long, curled Body length: 10.4 mm. setae on posterior (straight setae); epimeral plate 3 Gnathopod 1 (Fig. 20A, C): coxal plate bearing with blunt posterodistal corner (acute); and urosomites three setae and two setae on anterior and posterior flat (elevated). margins, respectively; basis with setae on anterior and posterior margins; propodus oval, palm with four GAMMARUS PISINNUS SP. NOV. (FIGS 22–27) spines on posterior margin; dactylus with one seta on outer margin. Material examined Gnathopod 2 (Fig. 20B, D): coxal plate bearing Holotype (IZCAS-I-A1254-1), male, 8.6 mm, Huo ′ ′ three setae and one seta on anterior and posterior Spring (36°18 N, 111°48 E), altitude 580 m, Hongdong margins, respectively; basis with short setae on ante- County, Linfen City, Shanxi Province, China, rior and posterior margins, posterodistal corner with 31.v.2012, collected by J. Li and Z. Chen. Paratypes short setae; propodus subrectangular, palm margin (from IZCAS-I-A1254-2 to IZCAS-I-A1254-12), six with four spines on posterodistal corner, bearing males and five females, same data as holotype. simple setae along anterior and posterior margins; dactylus with one seta on outer margin. Other material Pereopods 3, 4 (Fig. 21A, B): with shorter setae on Six males and six females (from IZCAS-I-A1227-1 to posterior margin than those of male. IZCAS-I-A1227-12, voucher number 1227), Queen Pereopods 5–7 (Fig. 21C–E): similar to those of Spring (36°41′N, 112°32′E), altitude 1034 m, Qinxian male. County, Shanxi Province, China, 7.v.2012, collected Uropods 1–3 (Fig. 19L–N): uropods 1 and 2 similar by J. Li and Z. Chen. Five males and five females to those of male. Uropod 3 peduncle with one spine (from IZCAS-I-A1229-1 to IZCAS-I-A1229-10, vou- accompanied by one seta on surface and four distal cher number 1229), Nianziguan Spring (37°58′N, spines; inner ramus about 1.4 times as long as pedun- 113°52′E), altitude 370 m, Yangquan City, Shanxi cle, reaching 0.6 times the length of outer ramus, with Province, China, 9.v.2012, collected by J. Li and Z. two spines accompanied by plumose setae on lateral Chen. Ten males and six females (from IZCAS- margin; article 1 of outer ramus with three pairs of I-A1216-1 to IZCAS-I-A1216-16, voucher number spines accompanied by simple setae on outer margin; 1216), Chitougou Spring (35°28′N, 112°32′E), altitude terminal article longer than adjacent spines. 581 m, Beiliu Town, Yangcheng County, Shanxi Prov- Telson (Fig. 21F): cleft, each lobe with two clusters ince, China, 2.v.2012, collected by J. Li and Z. Chen. of setae and one spine accompanied by two setae on Seven males and five females (from IZCAS-I-A1221-1 surface, bearing four or three distal spines accompa- to IZCAS-I-A1221-12, voucher number 1221), Xin’an nied by setae. Spring (36°22′N, 113°23′E), altitude 919 m, Lucheng

Figure 22. Gammarus pisinnus sp. nov., holotype, male. A, head; B, antenna 1; C, aesthetascs of antenna 1; D, antenna 2; E, calceoli of antenna 2; F, upper lip; G, lower lip; H, left mandible; I, incisor of right mandible; J, maxilla 1; K, palp of right maxilla 1; L, maxilla 2; M, maxilliped.

Figure 23. Gammarus pisinnus sp. nov., holotype, male. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 24. Gammarus pisinnus 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 7.

Figure 25. Gammarus pisinnus sp. nov., male, A–K; female, L–N. A, epimeral plate 1; B, epimeral plate 2; C, epimeral plate 3; D, urosomites (dorsal view); E, pleopod 1; F, pleopod 2; G, pleopod 3; H, uropod 1; I, uropod 2; J, uropod 3; K, telson; L, uropod 1; M, uropod 2; N, uropod 3.

Figure 26. Gammarus pisinnus sp. nov., female. A, gnathopod 1; B, gnathopod 2; C, propodus of gnathopod 1; D, propodus of gnathopod 2.

Figure 27. Gammarus pisinnus sp. nov., female. A, pereopod 3; B, pereopod 4; C, pereopod 5; D, pereopod 6; E, pereopod 7; F, telson; G, oostegite of gnathopod 2; H, oostegite of pereopod 3; I, oostegite of pereopod 4; J, oostegite of pereopod 5.

County, Shanxi Province, China, 5.v.2012, collected by Gnathopod 1 (Fig. 23A, C): coxal plate bearing four J. Li and Z. Chen. setae on anterior margin and one seta on posterior margin; basis with setae on anterior and posterior Etymology margins; carpus about 1.3 times as long as wide, The specific name is from the Latin pisinnus (little), about 0.6 times as long as propodus, posterior margin indicating the small size of this species; adjective. bearing short setae; propodus oval, palm with one medial spine and 11 spines on posterior margin and Diagnosis facial surface; dactylus with one seta on outer margin. Eyes reniform; merus to carpus of pereopod 3 with Gnathopod 2 (Fig. 23B, D): coxal plate bearing long, straight setae on posterior margin; epimeral three setae on anterior margin and one seta on pos- plates 2, 3 with few seta on posterior margin; inner terior margin; basis with long setae on anterior and ramus of uropod 3 reaching about 0.6 times length of posterior margins, posterodistal corner with short outer ramus. setae; carpus about 1.5 times as long as wide, about 0.6 times as long as propodus, bearing six clusters of Description setae along ventral margin, two clusters of setae on Holotype: male dorsal margin; propodus palm ovate, palm margin Body length: 8.6 mm. with one medial spine and five spines on lateral Head (Fig. 22A): eyes reniform, inferior antennal posterodistal corner; dactylus with one seta on outer sinus deep, lateral cephalic lobe nearly straight. margin. Antenna 1 (Fig. 22B, C): peduncular articles 1–3 in Pereopod 3 (Fig. 24A, F): coxal plate bearing two length ratio 1.0:0.9:0.4, with distal setae; flagellum setae on anteroventral margin and one seta on pos- with 28 articles, most with aesthetascs; accessory terior margin; basis elongate, with setae along pos- flagellum with four articles; both primary and acces- terior margins; merus with long, straight setae on sory flagella with short distal setae. posterior margin; carpus to propodus with groups Antenna 2 (Fig. 22D, E): peduncular articles 3–5 in of spines accompanied by long setae on posterior length ratio 1.0:2.8:2.6, peduncular articles 4, 5 with margin; dactylus with one plumose seta on posterior clusters of lateral and medial setae; flagellum with 12 margin, and two setae at hinge of unguis. articles; calceoli present in articles 1–7. Pereopod 4 (Fig. 24B, G): coxal plate excavated, Upper lip (Fig. 22F): ventral margin rounded, bearing two setae on anterior margin and three setae bearing minute setae. on posterior margin; basis with setae on posterior Mandible (Fig. 22H, I): left mandible incisor with margin; merus with straight setae on posterior five teeth; lacinia mobilis with four teeth, spine row margin; carpus to propodus with groups of spines with six pairs of plumose setae along ventral margin; accompanied by setae on posterior margin; dactylus palp articles 1–3 in length ratio 1.0:2.0:1.8; article 2 with one plumose seta on posterior margin and two armed with 12 marginal setae; article 3 with six setae at hinge of unguis. A-setae and four B-setae, plus 16 D-setae and five Pereopod 5 (Fig. 24C, H): coxal plate bearing one E-setae apically; incisor of right mandible with four seta on anterior margin and two setae on posterior teeth, lacinia mobilis bifurcate, with small teeth. margin; basis with four spines on anterior margin, Lower lip (Fig. 22G): inner lobes lacking, outer anterodistal corner with one spine, posterior margin lobes covered with thin setae. with a row of eight setae; merus to propodus with Maxilla 1 asymmetrical (Fig. 22J, K), left inner groups of spines on anterior margin; dactylus with plate with 18 plumose setae on medial margin; outer one plumose seta on posterior margin and two setae plate with 11 robust, serrated apical spines, each at hinge of unguis. spines with small teeth; article 2 of palp with nine Pereopod 6 (Fig. 24D, I): coxal plate bearing one seta slender spines and one seta apically; article 2 of right on posterior margin; basis elongate, with three setae palp with five stout spines, one stiff seta, and one and four spines on anterior margin, anterodistal slender spine. corner with two spines, posterior margin dwindling Maxilla 2 (Fig. 22L): inner plate with 15 plumose distally, with a row of eight setae; merus to propo- setae in an oblique row; inner and outer plates with dus with groups of spines on anterior margin, propodus long setae apically. with two groups of setae on posterior margin; dactylus Maxilliped (Fig. 22M): inner plate with three stout with one plumose seta on posterior margin and two apical spines and one subapical spine, some plumose setae at hinge of unguis. setae along ventral margin; outer plate bearing a row Pereopod 7 (Fig. 24E, J): coxal plate bearing one of blade-like spines and four plumose setae apically; seta on anterior margin and three setae on posterior palp article 4 hooked, with a group of setae at hinge margin; basis with three setae and three spines on of unguis. anterior margin, anterodistal corner with one spine,

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 170, 591–633 DIVERSIFICATION OF GAMMARUS SPECIES 631 posterior margin with a row of eight setae; merus posterior margins; propodus oval, palm with five to propodus with groups of spines on anterior margin, spines on posterior margin; dactylus with one seta on propodus with two groups of setae on posterior outer margin. margin; dactylus with one plumose seta on posterior Gnathopod 2 (Fig. 26B, D): coxal plate bearing margin and two setae at hinge of unguis. three setae and one seta on anterior and posterior Coxal gills: coxal gill of gnathopod 2 and gills margins, respectively; basis with setae on anterior of pereopods 3–5 a little shorter than bases; gill of and posterior margins, posterodistal corner with pereopod 6 more than half length of basis; gill of short setae; propodus subrectangular, palm margin pereopod 7 smallest, less than half of basis. with four spines on posterodistal corner, bearing Epimeral plates (Fig. 25A–C): plate 1 ventrally simple setae along anterior and posterior margins; rounded, bearing three setae on anteroventral dactylus with one seta on outer margin. margin, two setae on posterior margin; plate 2 with Pereopods 3, 4 (Fig. 27A, B): with shorter setae on two spines on ventral margin, one seta on posterior posterior margin than those of male. margin, posterodistal corner subacute; plate 3 with Pereopods 5–7 (Fig. 27C–E): similar to those of three spines on ventral margin, one seta on posterior male, but a little stout. margin, posterodistal corner acute. Uropods 1–3 (Fig. 25L–N): uropods 1 and 2 similar Pleopods 1–3 similar (Fig. 25E–G), peduncles with to those of male. Uropod 3 peduncle with one spine two retinacula accompanied by one or two setae; outer accompanied by one seta on surface and three distal ramus slightly shorter than inner ramus, both rami spines; inner ramus about 1.1 times as long as pedun- fringed with plumose setae. cle, reaching 0.5 times the length of outer ramus, Urosomites 1–3 (Fig. 25D): urosomite 1 with two− with one spine and plumose setae on lateral margin; two−two spines accompanied by setae on dorsal article 1 of outer ramus with three pairs of spines margin. Urosomite 2 with two−two−two spines accompanied by simple setae and plumose setae on accompanied by setae on dorsal margin. Urosomite 3 outer margin; terminal article longer than adjacent with two spines accompanied by setae on each side spines. and one cluster of setae on dorsal margin. Telson (Fig. 27F): cleft, each lobe with one or two Uropods 1–3 (Fig. 25H–J): uropod 1 peduncle with setae and one spine accompanied by one seta on one basofacial spine, two spines on outer margin, one surface, bearing four distal spines accompanied by spine on inner margin, two and one spines on outer two setae. and inner distal corners, respectively; outer ramus Oostegite (Fig. 27G–J): oostegite of gnathopod 2 with one spine on inner margin; inner ramus with broad, with marginal setae, oostegites of pereopods 3 two spines on inner margin; both rami with five and 4 elongate, oostegite of pereopod 5 smallest. terminal spines. Uropod 2 short, peduncle bearing one spine on outer margin and one spine on inner Habitat margin, with one distal spine on each corner; outer Gammarus pisinnus sp. nov. has been found in south- ramus with one spine on each margin; inner ramus ern parts of the Taihang Mts. All known individuals with two spines on inner margin. Uropod 3 peduncle have been collected from springs. Many springs in with one spine accompanied by two setae on surface this area have disappeared or diminished because of and four distal spines; inner ramus about 1.6 times as groundwater exploitation. As a consequence, some long as peduncle, reaching 0.6 times the length of endemic species have suffered population losses or outer ramus, with two spines on inner margin accom- become extinct, and efforts should be made to con- panied by plumose setae, bearing two apical spines serve this vulnerable habitat. accompanied by simple setae; article 1 of outer ramus with three pairs of spines on outer margin, both Remarks margins set with plumose setae; terminal article Gammarus pisinnus sp. nov. is similar to its sibling longer than adjacent spines. species G. clarus in epimeral plate 3 with acute pos- Telson (Fig. 25K): deeply cleft, as long as wide, each terior corner and both margins of uropod 3 with lobe with setae and one spine accompanied by two plumose setae. This new species differs from G. clarus setae on surface, bearing three distal spines accom- (character states in parentheses) in urosomites 1 and panied by setae. 2 flat (elevated); basis of pereopods 6 and 7 broad (proximally broad, distally narrow); telson with long Female, paratype (IZCAS-I-A1254-2) setae on the surface (with a few short setae). Body length: 6.8 mm. Gammarus pisinnus sp. nov. resembles G. Gnathopod 1 (Fig. 26A, C): coxal plate bearing two nekkensis, which was redescribed by Karaman (1989), setae and one seta on anterior and posterior margins, in the shape of epimeral plates and the ratio between respectively; basis with long setae on anterior and inner and outer ramus of uropod 3. Gammarus

KEY TO THE GAMMARUS SPECIES OF THE LÜLIANG AND TAIHANG MOUNTAINS 1. Pereopod 3 with short setae on posterior margin...... Gammarus shanxiensis – Pereopod 3 with long setae on posterior margin...... 2 2. Pereopod 3 with long, straight setae on posterior margin...... 3 – Pereopod 3 with long, curled setae on posterior margin...... 5 3. Eyes oval...... Gammarus benignus sp. nov. – Eyes reniform...... 4 4. Urosomites 1, 2 ﬂat...... Gammarus pisinnus sp. nov. – Urosomites 1, 2 weakly elevated...... Gammarus clarus 5. Outer margin of outer ramus in uropod 3 with only simple setae ...... Gammarus nekkensis – Both margins of outer ramus in uropod 3 with plumose setae...... 6 6. Epimeral plate 3 with blunt posterior corner...... Gammarus monticellus sp. nov. – Epimeral plate 3 with acute posterior corner ...... Gammarus incoercitus sp. nov.

pisinnus can be distinguished from G. nekkensis Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evo- (character states in parentheses) by the following lutionary analysis by sampling trees. BMC Evolutionary characters: eyes reniform (ovoid); pereopod 3 with Biology 7: 214. long, straight setae on posterior margin (long, curled Fišer C, Sket B, Trontelj P. 2008. A phylogenetic perspec- setae); and both margins of inner and outer rami of tive on 160 years of troubled taxonomy of Niphargus uropod 3 densely with plumose setae (outer margin of (Crustacea: Amphipoda). Zoologica Scripta 37: 665–680. outer ramus with long, simple setae only). Compari- Gong M. 2010. Uplifting process of Southern Taihang sons with other related species can be found in the Mountian in Cenozoic. PhD Thesis, Chinese Academy of Geological Science, Beijing, China. following key. Hillis DM, Mable BK, Larson A, Davis SK, Zimmer EA. 1996. Nucleic acids IV: sequencing and cloning. In: Hillis ACKNOWLEDGEMENTS DM, Moritz C, Mable BK, eds. Molecular systematics. Sun- derland, MA: Sinauer Associates, 321–328. We thank for Zhigang Chen for his help during field- Hou Z, Fu J, Li S. 2007. A molecular phylogeny of the genus work. We are grateful to four anonymous reviewers Gammarus (Crustacea: Amphipoda) based on mitochondrial for their valuable comments. This work was sup- and nuclear gene sequences. Molecular Phylogenetics and ported by grants from the National Natural Science Evolution 45: 596–611. Foundation of China to Z. H. (NSFC-31172049) and Hou Z, Li S. 2004. Three new species of Gammarus from the National Science Fund for Distinguished Young Shaanxi, China (Crustacea: Amphipoda: Gammaridae). Scholars to S. L. (NSFC-31025023). Journal of Natural History 38: 2733–2757. Hou Z, Li S. 2010. Intraspecific or interspecific variation: REFERENCES delimitation of species boundaries within the genus Gammarus (Crustacea, Amphipoda, Gammaridae), with Ba J, Hou Z, Li S. 2011. Modeling the distribution pattern description of four new species. Zoological Journal of the of freshwater Gammarus (Crustacea, Amphipoda) with Linnean Society 160: 215–253. Maxent. Acta Zootaxonomica Sinica 36: 837–843. Hou Z, Sket B, Fišer C, Li S. 2011. Eocene habitat shift Barnard JL, Dai AY. 1988. Four species of Gammarus from saline to freshwater promoted Tethyan amphipod (Amphipoda) from China. Sinozoologia 6: 85–112. diversification. Proceedings of the National Academy of Claramunt S, Derryberry EP, Remsen JV, Brumfield Sciences, USA 108: 14533–14538. RT. 2012. High dispersal ability inhibits speciation in a Karaman GS. 1989. One freshwater Gammarus species continental radiation of passerine birds. Proceedings of the (Gammaridea, Fam. Gammaridae) from China (Contribu- Royal Society B 279: 1567–1574. tion to the knowledge of the Amphipoda 189). Poljoprivreda Colson-Proch C, Morales A, Hervant F, Konecny L, I Sumarstvo 35: 19–36. Moulin C, Douady CJ. 2010. First cellular approach of Katoh K, Standley DM. 2013. MAFFT multiple sequence the effects of global warming on groundwater organisms: a alignment software version 7: improvements in performance study of the HSP70 gene expression. Cell Stress and Chap- and usability. Molecular Biology and Evolution 30: 772–780. erones 15: 259–270. Kelly DW, MacIsaac HJ, Heath DD. 2006. Vicariance and Daniels SR, Gouws G, Crandall KA. 2006. Phylogeogra- dispersal effects on phylogeographic structure and specia- phic patterning in a freshwater crab species (Decapoda: tion in a widespread estuarine invertebrate. Evolution 60: Potamonautidae: Potamonautes) reveals the signature of 257–267. historical climatic oscillations. Journal of Biogeography 33: Lanfear R, Calcott B, Ho SYW, Guindon S. 2012. 1538–1549. PartitionFinder: combined selection of partitioning schemes

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Relationship between Kimura two-parameter distances calculated for cytochrome c oxidase subunit I (COI) only and for combined mitochondrial (COI and 16S) genes. The slope of the regression is 1.2336. Figure S2. Maximum clade credibility chronogram inferred from a relaxed clock model based on mitochondrial [cytochrome c oxidase subunit I (COI) and 16S] data set. Node bars represent 95% posterior credibility intervals for nodes of interest. Vertical bars are used to designate clades.