Taxonomy of the spring dwelling amphipod Synurella ambulans ( Crustacea : Crangonyctidae ) in West Russia : with notes on its distribution and ecology

This study deals with taxonomic problems of the semi-subterranean crangonyctid amphipod Synurella ambulans (F. Müller, 1846), well-known from various freshwater habitats in Europe. The taxonomy of the species S. ambulans and the generic diagnosis for the genus Synurella are revised. A new synonymy is proposed: Synurella ambulans (F. Müller, 1846) = Synurella ambulans meschtscherica Borutzky, 1929, syn. nov. The affi nity with the related groups, distribution and ecology of the species are examined.

Some authors have considered integrating the genus Synurella into the genus Stygobromus, owing to the lack of clear morphological and geographical boundaries between these groups (Birstein 1948;Karaman 1974aKaraman , 1974b, or directly united them (Barnard & Barnard 1983). However, some researchers have expressed the opposite view (Holsinger 1977;Bousfi eld 1977). In our opinion, signifi cant morphological differences between these genera are primarily observed in the general body morphology. Moreover, the genus Synurella is not monophyletic according to Martynov (1931), who assigned all the Far Eastern-Siberian and one Alaskan species to the subgenus Eosynurella. The latter group differs markedly from the European taxa, except for Synurella dershavini Behning, 1928 and from the North American Synurella, by the pear-shaped gnathopod 2 propodi and the structure of uropod 3 with a strongly reduced terminal segment. It is possible that the biogeography of the genera Synurella and Stygobromus cannot be explained by a simplifi ed barrier-insulation approach proposed by several authors (Birstein 1948;Karaman 1974aKaraman , 1974b, but should apparently be explained by isolation through the existence of ancient seas, straits, and by evolutionary differences.
Synurella meschtscherica from the Meschtschera Lowland is now formally considered a junior synonym of S. ambulans. In our study we examined samples from the topotype locality of S. meschtscherica. We further include detailed distributional information based on morphological comparison of material from scattered localities in West Russia.

Taxonomic sampling
The specimens of S. meschtscherica were collected and studied from an extensive territory on the East European Plain (Fig. 1), ranging from the Baltic Sea basin (Pskov administrative area) to the Meschtschera Lowland in the east (Moscow, Ryazan, Vladimir areas) and in the south to the Central Russian Upland in the Oka River basin (Kaluga area) as far as the upper part of the Dniepr River basin (Bryansk area). The comprehensive collection of specimens was carried out mostly by the second author using a common hand net. The samples were preserved in 80% ethanol and are kept at the Institute of Biology and Soil Science (Vladivostok). Preserved material of S. ambulans at the Museum of Naturkunde (Berlin), collected and determined by M.L. Zettler from Kassow near Rostock (Mecklenburg-Vorpommern, Germany), was also used in this study for comparison of two species.

Morphology
All relevant morphological structures were examined and measured. To measure the body length, more precisely the distance along the dorsal side of the body from the base of the fi rst antenna to the base of  Müller, 1846) in western Russia. Legend: "empty" springs display undisturbed springs with a rich crenophilous fauna without S. ambulans. Literature data: Borutzky (1929), Chertoprud (2006aChertoprud ( , 2006b. Source: Blank Mapping Tools, Moscow (2009). the telson, the specimen was held in a ventro-dorsal position. A Lomo MBS-9 stereomicroscope with a scaled micrometer eyepiece was used to make this measurement and appendages were drawn using a Carl Zeiss NU-2 compound microscope equipped with a drawing device as modifi ed by Gorodkov (1961). The permanent preparations were made using polyvinyl lactophenol (PVL) and a methylene blue staining solution. A lens adapter LSN-23D by Zarf Enterprises for Nikon CoolPix 995 was used for digital photomicrography of the lateral cephalic lobes and appendages.
The term "defi ning angle" of the gnathopod propodi refers to the angle formed at the end of the palm and beginning of the posterior margin or the point at which the tip of the dactylus closes on the propodus (see Holsinger 1974). The nomenclature for setal patterns on segment 3 of the mandibular palp follows the standard introduced by Karaman (1970). The descriptive terminology follows a classifi cation system based on the homology concept proposed by Watling (1989). The following description was generated from a DELTA database (Dallwitz 2005) for the crangonyctid genera and species of the world.

Remarks
In our opinion, the Crimean form Synurella ambulans taurica Martynov, 1931, with a slightly extended basipodite of pereopod 7, is related to the southern species complex of S. intermedia and S. tenebrarum rather than to the nominative species. However, owing to the poor description it is diffi cult to reach a defi nite conclusion.  Starobogatov 1995: 192. -Chertoprud 2006a2006b: 382.
A distinctive feature of this species is a well-marked broad yellowish spot ( Fig. 2A) located on the dorsal surface of the head between eyes. The spot is discernible only in living animals.
BODY. (Fig. 2C) Stout, appendages shortened. Antenna 1 45% longer than antenna 2. Antenna 2 (Fig. 8B) fl agellum with 6 segments; calceoli absent. Gnathopod 1 (Fig. 7A), propodus palm transverse or scarcely sub-transverse with cutting margin acanthaceous, palm with 8 simple strong setae in two rows; dactylus, inner margin with setae. Gnathopod 2 (Fig. 7B), propodus palm with cutting margin acanthaceous, palm with 7 simple strong setae in two rows; dactylus, inner margin with setae. Pereopods 3-7 (FIG. 8C-E), dactyli about 45-50% length of corresponding propodi. Uropod 1 (Fig. 8G), inner ramus as long as peduncle. Uropod 2 (Fig. 8H) about 60 % as long as uropod 1. Telson (Fig. 8J) somewhat tapered distally, slightly elongate, 1.1x longer than broad, as long as uropod 3. Oöstegites 2-5 large, ovoid with long marginal setae. Karaman (1974a) pointed out a signifi cant variability in several morphological features for S. ambulans. However, in our analysis of individuals from the different parts of its range in Russia and Germany, we could not discover any signifi cant variance in the shape of the lateral cephalic lobes, epimera, uropods, telson or bases of pereopod 7 (Fig. 9). In adults we observed elongation of the pereopod 7 bases and the presence of many robust setae on the lower edge of the epimera 2-3. Ommatidia were larger in young animals but their number was smaller than the one in adults. We also noted a slight variation in the length of the antenna 1 50-55% length of body and 40-45% longer than antenna 2, and a considerable variation in the length of pereopods 3-7 dactyli (35-50% length to corresponding propodi). The number of segments in the fl agellum of antenna 1 equals 12-16. The specimens from Kholodny brook, near Yezerische Lake (Russia, Pskov area, Pustokshinsky) have a slightly different setation pattern of maxilliped palp segments 3 and 4 (Fig. 4L), but are otherwise indistinguishable. Borutzky (1929: 32) adduced several distinctive characters which, in his opinion, were suffi cient to distinguish S. a. meschtscherica from S. a. ambulans: relative length of the both antennae, the stronger armament of mouthparts (viz., presence of scopiform bundles of setules on palpi of mandibles and maxilliped, presence of molar setae), armament of uropod 3 peduncle, the shape and armament of telson. After analyzing Borutzky's description, we are convinced that he has mixed details (p. 33) of males and females without explanation: the cited characters of the antenna 2 and the gnathopods belong to the female, while the telson characters are typical of the male. Moreover, Borutzky (loc. cit.) compared his own "mixed" description to the incomplete description by Schäferna (1922), who also depicted the female's telson without indication of gender. Borutzky (loc. cit.) evidently did not have Müller's (1846) original description at hand, in which the latter explains why he attributed subspecies status to his specimens.

Remarks
The comparison of the material of S. ambulans from Russia, previously identifi ed as Synurella meschtscherica, with that from Germany revealed no morphological differences between them. Comparison of the variability of the original samples with species descriptions by Müller (1846), Schäferna (1922) and Borutzky (1929) showed that both species are identical. We therefore consider Synurella ambulans meschtscherica Borutzky, 1929 a junior synonym of nominative S. ambulans (F. Müller, 1846).
The taxonomic status and geographic distribution of previously described forms of S. ambulans are in need of a substantial revision. In our opinion the complex classifi cation of S. ambulans is caused by: 1) a poor fi rst description of the species by F. Müller and 2) a relatively wide distribution of the genus in Europe. It is possible that S. ambulans, ranging widely in Europe and Asia with signifi cant variability reported by some authors (see above), is actually a series of several cryptic species.
A few discrepancies were found in the comparison with the original description. Borutzky (loc. cit.) reported the body length of individuals within the range of 6-12 mm for mature specimens without an indication of the method of measurement. Our largest individual has a body length of 6.0 mm. We have also studied the samples from Velikodvorskye springs of Ryazan area, previously also explored by Borutzky, where females up to 5.0 mm body length were found. Borutzky (1929: 32) also found a somewhat larger number of segments of the fl agellum of the antenna 1, 18-24 (males) and 16-22 (females), and reported on eyeless individuals which are absent in our collections. However, the observed variability was not documented by this author, eyeless individuals were not described and had not been given a special status. The inaccuracy of Borutzky's description confi rmed our doubts about the validity of S. meschtscherica and convinced us that only one form of Synurella is present in the Meschtschera Lowland.
Synurella ambulans dwells in various springs, stagnant parts of the rivers and brooks connected with the ground outlets of subterranean waters, frequently associated with the asellid isopod Asellus aquaticus (Linnaeus, 1758). A characteristic features of all microhabitats are their stagnant or very slowly fl owing waters, not exceeding 0.1 m/sec; a water temperature generally ranging between 2.0 and 16.0 °C, a low oxygen concentration of 3.0-9.0 О 2 mg/l, a рН between 5.0-8.0 and low mineralization not higher than 197.5-353.1 mg/l (once 510.0 mg/l) (Nesemann et al. 1995;Giginyak & Moroz 2000;Chertoprud 2006a). Springs are often covered with Lemna and Hydrocharis, or densely grown with Elodea and Fontinalis; bottoms are composed of detritus, sand, mud, snags and leaf litter. Dendrocometes paradoxus Stein, 1852 (Protozoa, Infusoria, Suctoria) is a common ectoparasite on the coxal gills of S. ambulans (see Taylor & Sanders 2001).
The rare fi ndings of S. ambulans in a number of a small fl oodplain lakes in the spring could be explained by the fl ood drift. However, most interesting is the accidental discovery (by DP) of a mass congestion of S. ambulans on the shallows of a large lake in the Velikaya River basin (Pskov area) in winter. It is possible that these crustaceans can survive adverse winter conditions by "warming up" near oozing from the bottom fontanels.

Comments on biogeography of Synurella species complex
The biogeography of any group is closely linked with its phylogenetic relationships (Holsinger 1986), which in turn is related to a system of trustworthy diagnostic features. It is well-known that the taxonomy of the family Crangonyctidae is based largely on the structure of uropod 3, although the plasticity of this character had already been discussed (Bousfi eld 1983) and a recent molecular phylogeny confi rms this (Hiwatari et al. 2011). The latest results of 18S phylogeny also revealed an ancient radiation of Synurella with a paraphyletic relationship to the North American and European groups (Kornobis et al. 2011).
Morphologically, the family Crangonyctidae is not homogeneous and can be tentatively divided into two groups of uneven size, but equivalent in terms of their "biogeographical weight". The fi rst group includes taxa with the "free coxal plates 1-4" or commonly diagnosed as shallow coxae, when their width is greater than, or equal to, the height (this group includes: Bactrurus, Siberian Stygobromus and the majority of the North American Stygobromus). Two poorly described Eurasian species -Stygobromus apscheronicus (Derzhavin, 1945) and Stygobromus kazakhstanicus Kulkina, 1992 -occupy an intermediate position but they might belong to this group, because of their shallow coxal plates 3 and 4. The second large group, however, consists of species with deep coxal plates and is distinguished by a coxal plate 4 with excavation. This group includes the North American and European Crangonyx, Synurella, Lyurella hyrcana Derzhavin, 1939, Stygonyx courtneyi Bousfi eld & Holsinger, 1989, Amurocrangonyx arsenjevi (Derzhavin, 1927 and Palaeogammarus. The same character was used previously by Bousfi eld (1977) and Holsinger (1986) in a hypothesized phylogenetic relationship among Crangonyctidae. They ascribed a plesiomorphic state for the deep coxae and an apomorphic state for shallow or reduced coxae in overall size (see Holsinger 1986: 90). The front edge of the Crangonyctidae head is rather diverse in morphology (Holsinger 1977) and can be roundish (Fig. 9) or characterized by the presence of the inter-antennal lobe and inferior sinus. However, this feature apparently may not be appropriate for phylogenetic analyses, as we have observed non-uniform gradation from the roundish form (S. ambulans) with intermediate form (S. derzhavini) to the expressed "sinusoidal form" (S. osellai) (see Sidorov et al. 2012). Meanwhile, we have concluded that both features of general body morphology are most important for separation of heterogeneous phyletic groups in future testing of biogeographic hypotheses.

Taxonomic boundaries and eco-geographic distribution of Synurella ambulans
Synurella ambulans has a large distribution in the lowlands of northern Europe, ranging from NW to NE Europe and possibly reaching the Black Sea area (Dedju 1967). Previously, from pieces of Baltic amber dated as far back as the Eocene, several forms of Synurella have been described as being closely related to the southern species S. intermedia, because of the somewhat extended pereopod 7 bases and the non-acute posteroventral epimeral corners (see Coleman 2004: 3). Perhaps the northern distribution of S. ambulans may be a relatively recent phenomenon, after the retreat of the glaciers in the early Holocene. However, we do not know the southern and western boundary of the distribution of this species. The fi ndings of S. ambulans in Southern Europe and Asia Minor indicate that its range is discontinuous. This assumption is questionable and requires additional verifi cation. In our view, all references to S. ambulans in Asia Minor (Ruffo 1974;Karaman 2003;Ustaoğlu et al. 2004) as well as in Southern Europe (Bonacina et al. 1992;Stoch & Dolce 1994;Pezzoli 2010) are ambiguous and should be recompared carefully with S. ambulans from other areas.
Synurella ambulans is absent from a number of intact springs, unaffected by anthropogenic stress (Fig. 1). These springs were almost always rich in crenophilous fauna. As noted by Giginyak & Moroz (2000), Synurella ambulans inhabits springs with water close to melt water in physical and chemical properties. The natural tolerance of the studied amphipod specimens of S. ambulans was limited to low concentration of oxygen and mineralization (see Giginyak & Moroz 2000: 82). Previously, a similar relationship between life in springs and low water hardness of 1.2-1.6° dGh (= 214.1-285.6 mg/l) was noted for the Far Eastern Amurocrangonyx (Birstein & Levanidov 1952). It is interesting that S. ambulans apparently has a feature that can distinguish it from the other species of the genus, namely a yellowish spot (url: www.biospeleo.ru/S_ambulans.htm) which is the hypertrophied digestive gland. Previously, the observation of a yellowish (orange) spot for the species was mentioned by Müller (1846), Stebbing (1906), Borutzky (1929) and Boets et al. (2010). The hypertrophied digestive gland (HDG) is characteristic also of several deep-sea marine amphipods, e.g. Eurythenes gryllus Lichtenstein, 1822 where it performs the leading role in the metabolic response of the defense antioxidant system (Camus & Gulliksen 2004). In subterranean amphipods the HDG has also been observed in Amurocrangonyx.