Northernmost discovery of Bathynellacea (Syncarida: Bathynellidae) with description of a new species of Pacificabathynella from Alaska (USA)

Abstract A new species of the genus Pacificabathynella Schminke and Noodt, 1988 is described from groundwaters of Alaska (USA). This is the first record of Bathynellacea Chappuis, 1915 from the far north of America. Pacificabathynella has hitherto been known only from the states of California (one species) and Montana (three species). Pacificabathynella and Paradoxibathynella Serban, 2000 are the only genera that show sexual dimorphism in thoracopod VI. Pacificabathynella yupik sp. nov. has several unique features within the genus: antenna eight-segmented; antennule only slightly longer than the antenna; the setal formula of the maxilla (7/3/7/5); without seta on the endopod of the male thoracopod VIII; five spines on the endopod of the uropod and the endopod as long as the sympod. The new species further shows slight differences in the antennula, pars molaris of the mandible, in thoracopod I–VII and in thoracopod VIII of males and females with the other species of the genus. Sequences of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) of five specimens of the new species were obtained to complete the morphological description with molecular data.


Introduction
The Family Bathynellidae Grobben, 1905 in North America includes only seven known species belonging to two genera: Bathynella Vejdovsky, 1882, with three species (one in Colorado and two others in California) and Pacificabathynella Schminke and Noodt, 1988, with four species, one in San Francisco, California and three in Montana ).
This family is widespread throughout the world, with 28 genera and 102 species (Camacho 2015) but is poorly known in North America. Current taxonomy of the genus Bathynella is not satisfactory, and includes more than 50 species and subspecies (Camacho 2006) distributed throughout the world, but few have been properly studied.
In 1988, Pacificabathynella was the only known genus with sexual dimorphism in thoracopod VI of the male. Serban (2000) described a new genus from Japan, Paradoxibathynella, which also has similar sexual dimorphism, to include Bathynella yezoensis (Uéno, 1954) considered as belonging to Pacificabathynella by Schminke andNoodt, 1988. Serban (2000) created the new tribe Pacificabathynellini to include the two genera with sexual dimorphic thoracopod VI.
The new species described in this paper undoubtedly belongs to the genus Pacificabathynella (see Table 1).
Normally, unique combinations of the morphological characters can define genera and species, but individually these characters do not seem to delineate species in this special groundwater crustacean group. Due to extreme morphological simplification and convergence in Bathynellacea Chappuis, 1915(Camacho et al. 2013, in some cases morphology alone is not enough to identify closely related species and this causes difficulties in the generic assignment of species as well. So, it now appears essential to obtain DNA sequences for authentic comparison of closely allied species. This is particularly true in the case of the Bathynellidae, where a combined molecular and morphological approach is strongly recommended for any future taxonomic research. In this paper we have followed both these approaches to characterise Pacificabathynella yupik sp. nov. from the USA. The sequences of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) were obtained from five specimens of the new species to supplement the morphological description and also have reference characters in our library of genes that allow future comparisons. The nuclear 18S rRNA is usually used to find relationships Table 1. Characteristics of the two genera of the Tribe Pacificabathynellini Serban, 2000. Pacificabathynella Schminke and Noodt, 1988and Paradoxibathynella Serban, 2000. Abbreviations: A.I, antennule; A.II, Antenna; Th, thoracopod. (Modified after  between genera of a family. The small subunit (SSU) 18S rRNA gene is one of the most frequently used genes in phylogenetic studies and an important marker for random target PCR in environmental biodiversity screening (Chenuil, 2006), but unfortunately we failed to obtain a long fragment of 18S. With this and other recent discoveries from glaciated western Montana (i.e. Camacho, Stanford, et al. 2009), the true diversity of the group in North America has begun to emerge. For the first time a species is found at a northernmost point in the boreal hemisphere, above latitude 60 (60.34520°N). Antrobathynella stammeri stammeri (Jakobi, 1954) was found in the River Lathkill, Derbyshire, England. (54.1896195°N/-2.4015749 W) and Baikalobathynella magna (Bazikalova, 1954) was found in the Lake Baikal, Angara river bassin (53.5600973°N/108.1646634°E), both only slightly above latitude 50.

Material and methods
The specimens studied come from one sample collected in a locality in western Alaska (USA): gravel bar, Kwethluk River, a tributary of the Kuskokwim River.

Study area
The Kwethluk River is a fifth-order tributary to the Kuskokwim River within the Yukon Delta National Wildlife Refuge, Alaska, USA. The Kwethluk originates in the Eek and Crooked Mountains and flows northwest for 222 km across an expansive tundra plain underlain by permafrost (Johnson and Hartman 1969). The catchment is 3367 km 2 . The region has a subarctic climate with temperatures ranging from above 12°C in summer to -12°C as a winter average (Alt 1977). The river supports a forested riparian corridor on expansive alluvial flood plains underlain by shallow alluvial aquifers that moderate temperatures and prevent permafrost. Woody vegetation consists of willow (Salix spp.), alder (Alnus spp.), cottonwood (Populus sp.), and spruce (Picea glauca) stands, with substantial influence by beaver (Castor canadensis) herbivory and dam building (Mouw et al. 2013). The Kwethluk becomes ice-free in May and freezes again in November (Roettiger et al. 2004). The climate is dry, averaging approximately 50 cm/ year of precipitation, with most of it falling as rain in late summer. Downstream from the head waters, the mainstem Kwethluk is a low-gradient wandering river with an anastomosing, sometimes anabranching network of primary, secondary, and tertiary channels with many flood channels and lateral habitat typesa shifting habitat mosaic (after Stanford et al. 2005).

Groundwater sampling methods
The sample was collected in wells. Groundwater wells were installed using a hollow 5.0 cm diameter metal piezometer pounded into the substrate with a centre rod for at least one metre. Then, the centre rod was removed and a 3.8 cm piece of PVC was installed with slits cut into the lower half with a circular saw. Once the PVC was fitted into the hole, the metal piezometer was pulled out leaving only the PVC in the substrate.
During sampling events, we lowered a tube into the PVC and water was pumped with a hand-operated bladder (peristaltic) pump and pumped for 10 minutes through a catch net (100 μm) suspended in a 20 litre bucket. Pumping continued for 10 minutes regardless of the clarity of the water. Then, the net was inverted and washed so all organisms were collected into the bucket. Finally, the sediment, detritus, and organisms pumped from the wells were poured into Nalgene bottles and preserved with 95% ethanol. The specimens used for morphological and molecular study were stored in alcohol (95%). The specimens were used for molecular study after they were frozen at −20°C, in 500 μl of an appropriate buffer.
The morphological and molecular descriptions are based on the type series.

Morphological study
In all, 37 specimens were used for the morphological study: 32 females and five males collected on 24 June 2012 in well #5, gravel bar, Kwethluk River, a tributary of the Kuskokwim River. They constitute the type series of the new species described herein. A complete dissection of all anatomical parts of all specimens was done and kept as permanent preparations (special metal slides, glycerine gelatine stained with methylene blue as the mounting medium). Anatomical examinations were performed using an oil immersion lens (100×) of a Zeiss interference microscope with a drawing tube. This material was deposited in the Collection of Arthropoda not Insecta of the Museo Nacional de Ciencias Naturales, Madrid (MNCN).
We used the terminology proposed by Serban (1972, and his subsequent papers) for the mandible and male thoracopod VIII. Serban worked in depth on the family Bathynellidae , Serban 1989a, 1989b, revised and described many genera, and we think that his terminology is the most accurate and intuitive.

Specimens collection for DNA extraction
Five specimens, which are part of the type series, were used for DNA extraction in this study. This type material, the DNA extracted from the five specimens, is deposited in the Tissues and DNA Collection of the Museo Nacional de Ciencias Naturales, Madrid (MNCN). Voucher numbers are shown in Table 2.
To further examine the phylogenetic relationships among bathynellids, we used partial DNA sequences of the mtDNA gene COI (508 base pair, bp). COI sequences were obtained from all individuals. DNA extraction, amplification, and sequencing Whole specimens were placed fully immersed in 0.5 ml digestion buffer (Gilbert et al. 2007), and incubated overnight at 55°C with gentle agitation. The buffer consisted of 5 mM CaCl 2 , 2% sodium dodecyl sulphate (SDS), 40 mM dithiotreitol (DTT), 250 mg/ml proteinase K, 10 mM Tris buffer pH 8, 2.5 mM EDTA (Ethylene-Diamine-Tetra-Acetic acid) pH 8.0, and 10 mM NaCl (final concentrations). After incubating, nucleic acids were extracted from the digestion buffer using a QIAquick PCR purification kit (QIAGEN), (Alda et al. 2007).
A 508 bp region of the COI gene was amplified with the primers C1-J-1718 (5ʹ-GGAGGATTTGGAAATTGATTAGTTCC-3ʹ) and HCO2198 (5ʹ-TAAACTTCAGGGTGACCAA AAAATCA-3ʹ) (Folmer et al. 1994;Simon et al. 1994). Three microlitres of the DNA solution were used as a template. Other components of the 25 μl PCR reaction were: 1× of the corresponding buffer (75 mM Tris HCl, pH 9.0; 50 mM KCl and 20 mM (NH 4 ) 2 SO 4 , 2 mM MgCl 2 ), 10 mM dNTPs mix, 0.1 μM of both primers, 0.02% BSA, and 0.125 units AmpliTaq Gold® DNA Polymerase (Applied Biosystems). Five microlitres of PCR products were electrophoresed through a 1.5% agarose gel and visualised with SYBR Safe™ DNA Gel Stain (Invitrogen) under ultraviolet light. PCR products were purified by treatment with ExoSAP-IT (USB Amersham, Buckinghamshire, UK) and incubated at 37°C for 45 min, followed by 80°C for 15 min to inactivate the enzyme. Purified PCR product was then used to sequence in both directions using the BigDye Terminator v3.1 sequencing kit (Applied Biosystems Inc., Foster City, USA) in a 10 μl volume, containing 15-20 ng of purified product and 3 pmol of primer. To verify that the sequences obtained came from a bathynellacean, they were compared with sequences from GenBank using Blast (Altschul et al. 1997). The alignment of all bathynellacean COI gene sequences generated in our lab was performed and edited manually using MEGA 5.0 (Tamura et al. 2011). Fine adjustments were made by eye, as the COI does not generally have single gaps. All sequences were submitted to GenBank (see Table 2 for the GenBank Accession Number).
In 1988, Pacificabathynella Schminke and Noodt, 1988 was the only known genus with sexual dimorphism in thoracopod VI of the male. Schminke and Noodt (1988) proposed that the species Bathynella yezoensis Uéno, 1954, recorded from wells in Hokkaido (Japan), must belong to the genus Pacificabathynella, mainly because of the type of dimorphism, which is similar to the dimorphism seen in Pacificabathynella sequoiae Schminke andNoodt, 1988. However, Serban (2000) described a new genus from Japan, Paradoxibathynella, which has the same type of sexual dimorphism. He considered, after studying all the characters of Pacificabathynella yezoensis (Uéno, 1954), that this species belonged to Paradoxibathynella Serban, 2000 and not to Pacificabathynella (see Serban 2000 and for a detailed discussion). Serban (2000) created the new tribe Pacificabathynellini, which includes the two genera exhibiting sexual dimorphism on thoracopod VI.
The new species described in this paper belongs to the genus Pacificabathynella (see Table 1).
Family BATHYNELLIDAE Grobben, 1905 Pacificabathynella Schminke andNoodt, 1988 Amended genus diagnosis (after Schminke andNoodt 1988 and) Antennule 7-segmented and antenna 7-or 8-segmented. Mandible with two teeth on incisor process (pars incisiva), processus incisivus accessorius with one tooth and one seta-like tooth, pars molaris with two bidentated structures, parallel to main axis of the teeth, the most distal tooth very strong with apical denticles. Labrum with a small central protuberance at the distal edge. Thoracopod (Th) VI and VIII sexually dimorphic. Male Th VI with 3-segmented endopod, second segment broad and dilated with strong curved seta at outer margin; first segment also broader than usual. Female Th VI with 4segmented endopod and setal formula 1+0/0+1/0+0/2(1). Male Th VIII with three lobes around genital opening on coxa and with a tooth-like protuberance, prominence in the basipod, exopod and endopod 1-segmented. Female Th VIII bearing epipod; exopod and endopod 1-segmented, both with two or three apical setae. Endopod of uropod with four to eight strong spines along inner margin. Furcal rami with five spines.  Table 3. Body not very elongated; almost cylindrical, approximately nine times as long as wide; segments slightly widening towards posterior end. Head as long as wide. Pleotelson with one plumose dorsal seta on either side, similar to furcal rami. All drawings are of the holotype (male) except for Th VIII and one figure of Md, paragnath and Th VI that belong to the allotype (female).   Antennula (Figure 2A). Seven-segmented; length of first three segments similar to other four segments combined; segments four and five smaller than others and with similar length; sixth segment shorter than seventh that is the longest; inner flagellum  almost trapezoidal; setation as in Figure 2A; two aesthetascs on sixth and three on seventh segments. A.I slightly longer than antenna.
Labrum ( Figure 2C). With a small central protuberance at the distal smooth free edge, flanked by slight protrusions, most evident on ventral side and with a visible small denticle in one of these protrusions.
Paragnath ( Figure 2D, E). Short, almost square, with rounded distal part and very thick setulation on all surfaces on distal half.
Mandible ( Figure 2F and G). Palp with three segments, terminal segment ( Figure 2G) with two long and strong barbed claws, more or less cylindrical without expansions. Masticatory part ( Figure 2F, H): incisor process (pars incisiva) with two teeth; processus incisivus accessorius with one tooth and one long seta-like tooth; pars molaris with two dentate structures, like two crowns, parallel to main axis of teeth, the first (the nearest to processus incisivus accessorius) with consistantly three strong denticles and the other with small denticles, except the most distal which is a strong tooth.
Thoracopods I-VII ( Figures 3A-E, 4A-B). Sexual dimorphism present on Th VI. Th I ( Figure 3A) smaller than others; Th II ( Figure 3B) to V ( Figure 3E) similar in size and Th VII ( Figure 4B) a few longer than others. Th I without epipod; coxa with a long and strong plumose seta; basipod with two smoth setae and a tuft of long fine setules near base. Epipod present on Th II-VII, small in Th II-V, half the length of basipod; as long as the basipod on Th VI and VII. Exopod with one segment on all thoracopods, shorter than endopod in all cases, almost like the first two segments in Th I-III and Th VII and slightly longer than the first two segments in Th IV and V; with five barbed setae, two terminal, one dorsal and two ventral. Endopod with four segments in all thoracopods, setal formulae ( Figure 5A,B) with aberrant structure, with only three segments in endopod: first segment broader than usual, second segment broad and dilated bearing on outer margin strong medially curved seta, third segment small with two setae, one long and another shorter.
Male thoracopod VIII ( Figure 5A,B). Longitudinal axis of coxa and basipod form 25°a ngle. Penial region with frontal lobe, inner lobe, outer lobe. Frontal lobe with two lobules on distal end. Inner lobe more or less similarly developed as frontal lobe, distal region with projection claw (Prj) on internal side. Outer lobe with two well-developed lobules. Basipod very large, with rows of setules on inner side, with distal and very welldeveloped, crest-like protuberance and with one lateral-subdistal smooth seta and other distal. Endopod one-segmented, small, 1/4 length of exopod, with small denticles. Exopod well developed, longer than basipod, with five setae.
Pleopod I ( Figure 5D). Two segments, first segment with one very long plumose seta; second segment with seven setae, the three distal of different length and plumose.
Female thoracopod VIII of the allotype ( Figure 5C). Coxa with one small, barbed lateral seta; very large epipod, twice as long as basipod; endopod one-segmented, with two apical barbed setae of different length; exopod two times as long as endopod, with three setae, two apical, all smooth, but differing in length.
Pleotelson ( Figure 5F). With one long, plumose dorsal seta on either side near base of furca.
Furcal rami ( Figure 5F). Almost square, bearing five spines; dorsal spine similar to third, slightly longer than fourth and almost 70% length of second spine and first spine twice as long as second spine.

Variability
The observed variability affects the number of setae of the different segments of the endopods of the thoracopods I to III on females (see Table 4).

Discussion
Pacificabathynella and Paradoxibathynella are the only known Bathynellidae genera in the world that show sexual dimorphism in the endopod of Th VI. The transformations of Th VI in the male are similar in both genera. Table 1 shows the characteristics of both genera. The new species undoubtedly belongs to the genus Pacificabathynella as evidenced by several characters. Thoracopod VIII of the male of the new species has three lobes in the penial region with a protuberance or projection as a claw, whereas Paradoxibathynella has four lobes; the basipod has a crest-like protuberance, which is well developed in the new species, but it is lacking in Paradoxibathynella. The pars molaris of the mandible has two dentate structures in the new species, as opposed to six teeth in Paradoxibathynella. The coxa of the female Th VIII has one plumose seta in the new species, but three setae in Paradoxibathynella. The second segment of the first pleopod has seven setae in the new species, whereas there are eight or nine in Paradoxibathynella. The sympod of the uropod of the new species has five spines versus four in Paradoxibathynella, and five spines on the endopod versus four spines in Paradoxibathynella. In general, Paradoxibathynella has fewer spines and setae than Pacificabathynella.
Tables 5, 3 and 4 show the similarities and differences between the five species of the genus Pacificabathynella.
The new species, as we have seen, also shows affinities with P. kallispellensis. The sexual dimorphism is similar in all species and is only exhibited in Th VI and Th VIII. In males there are only three segments on the endopod, with the second segment broadly dilated, bearing on its outer edge a strong and curved medial seta. The first segment is also broader than usual, and the third segment is similar to the fourth segment of the female Th VI and Th VII of the male and female.

Remarks on the distribution of bathynellaceans in USA
So far, only 17 bathynellids species are known in North America, despite its vastness. Even the first finding (Texanobathynella bowmani Delamare Deboutteville et al., 1975) is relatively recent. To date, the family Parabathynellidae includes only 10 known species, belonging to five genera that inhabit the states of California, Virginia, Texas, and Montana (Califobathynella Cho, 1997, Californibathynella Camacho and Serban, 1998, Hexabathynella Schminke, 1972, Texanobathynella Delamare, Coineau et Serban, 1975and Montanabathynella Camacho, Stanford, et al., 2009). There are large areas to be sampled so presumably there are many new species that remain to be discovered. Until 2000 only four species of the family Bathynellidae were known, in two genera, Bathynella and Pacificabathynella from Colorado and California, respectively. We described in 2009 three species found in the state of Montana, extending the range of distribution of Pacificabathynella (see ) by over 1700 km. Now, the new species from Alaska widens the distribution range for this family 3000 km further north.
Many species found by different workers have not yet been formally described: Noodt (1974) reported bathynellids from California; Pennak and Ward (1985) reported bathynellids having been collected by their colleagues in the states of Montana, Wyoming, Colorado, Kansas, Oklahoma, Indiana, Ohio, and Georgia. Newell and Camacho have studied various populations of Montana, Texas, and Washington State, which have not yet been formally described, but some of them do belong to Pacificabathynella.