Description of a new species of Hobbsinella (Crustacea, Bathynellacea, Bathynellidae) from Colorado (USA) based on morphological and molecular characters

. A new species of Bathynellidae is described from Colorado (USA). Hobbsinella gunnisonensis Camacho & Taylor sp. nov. displays a unique combination of morphological characters including seven-segmented antenna lacking medial seta on exopod, antennule slightly longer than antenna, three-segmented mandibular palp, four articles on endopod of thoracopods I to VII and five spines on sympod and three spines on endopod of the uropods


Introduction
The crustacean family Bathynellidae Grobben, 1905 is widespread throughout the world, including 36 genera and 109 species (Camacho et al. 2021).This family has been poorly studied in North America (Camacho et al. 2018a), only nine species from three genera are known (Camacho et al. 2018b): three species of the genus Bathynella Vejdovsky, 1882 (from California and Colorado), five species of the genus Pacificabathynella Schminke & Noodt, 1988 (from California, Montana and Alaska) and one species of the genus Hobbsinella Camacho et al., 2018, from Texas.Bathynellidae show great morphological homogeneity and convergence due to the environmental pressures of the habitat where they live (groundwater).Therefore, character identification to differentiate species and genera is quite challenging.Molecular techniques can be particularly useful in providing additional information to help delimit within Bathynellidae.
1.The genus Bathynella isn't properly described.2. Many species have not been adequately described and need to be revisited and therefore comparisons are very difficult.3. Bathynella became, with time, the "catch-all" genus where most of the described Bathynellidae have been placed.4. So far, B. riparia Pennak & Ward, 1985 is the only species described for Colorado but needs revision.
In this paper, we describe a new species of the genus Hobbsinella originally from Texas.To support the morphological descriptions, we obtained sequences of mitochondrial (COI) and nuclear (18S) DNA from several specimens.With the description of Hobbsinella gunnisonensis Camacho & Taylor sp.nov.and its molecular characterization, the genus range is broadened in North America.Hobbsinella gunnisonensis sp.nov. is found in the West of the Continental Divide and H. edwardensis Camacho et al., 2018, is found East of the Continental Divide in a different habitat type and at a markedly lower elevation.We expand the distribution species of this genus, now known to span a range of more than 1300 km between type localities, from Texas to Colorado.
In the summer of 2018, field surveys of mixing zone and hyporheic waters were sampled with a Bou Rouch pump across the Colorado Rockies (Fig. 1A).Sites (n = 50) were stratified within USEPA HUC8 basins in the Southern Rockies Level IV Ecoregion (Fig. 1A), with specific locations determined by accessibility and feasibility of sampling.The Watershed Boundary Dataset from the U.S. Geological Survey -National Geospatial Program (2021) was used to delineate drainage basins.The map (Fig. 1) was generated in QGIS (ver. 3.20.1-Odense, 2021), with further processing in Affinity Designer (ver. 2.0.4, 2023).At each field site, three to four replicate samples were collected at haphazardly selected locations, at least 2 m apart and in substrate suitable for the sampling apparatus.For each sample, we used a Bou-Rouch GW pump, collecting 10 l of water which was filtered through a fine mesh aquarium net.Samples were preserved in 95% ethanol and kept in a cooler until transported to the laboratory for storage in a freezer.Samples were sorted in the laboratory (Bonwell et al. 2019) and bathynellaceans, found at only 2 of the 50 sites, were shipped from Colorado to the senior author of this paper (AIC) for morphological and molecular anaylyses.The 11 specimens of the new species described here were collected from the mixing and/or hyporheic zone of the Lottis Creek (eight specimens) and Spring Creek (three specimens) (Gunnison County), both sites are tributaries of the River Gunnison (Fig. 1A) (Table 2) The type locality is Lottis Creek off Forest Road 742, Gunnison County, Colorado.
Specimens used for morphological study were later fixed in 4% buffered formalin and stored in 70% ETOH.Specimens used for molecular study were directly frozen at -20°C, in 400 ml of distilled water or 0.5 ml digestion buffer.Five DNA extracts (one whole specimens and four abdomens, corresponding to four specimens of the type series and one of the additional material) of the new species are used in the molecular analysis (Tables 1-2).

Morphological study
Morphological descriptions are based on the holotype (female), type series (6 specimens) and additional material (three specimens).
For the morphological and molecular study of the new species, Hobbsinella gunnisonensis sp.nov., eleven females were used (see Tables 1-2 for specimen details, vouchers and GenBank numbers).The abdomen of six specimens were removed and used to extract DNA together with one whole specimen (Table 2).Eight specimens constitute the morphological type series of the new species described herein.A complete dissection of all appendages was performed and the resultant body parts were preserved as permanent slides (special metal slides, glycerine-gelatine stained with methylene blue and paraffin as mounting medium; see Perina & Camacho 2016).The morphological examination was performed using an oil immersion lens (at 1000 × magnification) with a Zeiss interference microscope.Drawings were done using a drawing tube, digitalised using a WACOM Tablet and retouched using Freehand and/or Adobe Illustrator drawing software.The material is deposited in the Arthropod collection of MNCN (ARTP/MNCN).The morphological terminology used throughout the text follows Serban (Serban 1972;Schminke & Noodt 1988).(2020).The samples were placed in 0.5 ml digestion buffer (Gilbert et al. 2007), and incubated overnight at 55°C with gentle agitation.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 incubation, nucleic acids were extracted from the digestion buffer using a Qiaquick PCR purification kit (QIAGEN) (Alda et al. 2007).

Abbreviations used in text and tables
Partial sequence of the COI gene were amplified with the primers C1-J-1718 and HCO2198 (Folmer et al. 1994;Simon et al. 1994) for all specimens.18S rRNA partial sequences were amplified in three fragments, using the primers 1F and 3R; 3F and 5R and 5F and 9R (Giribet et al. 1996).We used 3 µl of DNA extract for template.Other components of the 25 µl PCR reaction included 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).The PCR program consisted of an initial denaturation step of 95°C for 10 min, followed by 60 amplification cycles (95°C for 30 s, 45-49°C for 45 s and 72°C for 45 s) and a final elongation step of 72°C for 10 min.PCR were run on an Eppendorf Mastercycler gradient.PCR product (5 µl) was electrophoresed through a 1.5% agarose gel and visualized with SYBR SafeTM 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 products were sequenced in both directions using the BigDye Terminator ver.3.1 sequencing kit (Applied Biosystems Inc., Foster City, USA) in a 10 μl volume, containing 15-20 ng purified product and 3 pmol primer (Camacho et al. 2016).
DNA extractions have been deposited in the Tissues and DNA Collection of the MNCN.Voucher numbers of the seven specimens of the new species used in the molecular analyses are shown in Table 2.

Phylogenetic analysis
To explore the phylogenetic relationships within Bathynellidae, we use partial sequences of the mtDNA gene cytochrome oxidase 1 (COI) (509 bp) and the nuclear 18S rRNA (1071 bp) from a total of 31 specimens.These data sets include 11 clearly identified genera (Vejdovskybathynella, Paradoxiclamousella, Gallobathynella, Bathynella, Hobbsinella, Antrobathynella, Altainella, Pilbaranella, Fortescuenella, Anguillanella and Muccanella) and 18 well identified species (Table 1).We used Iberobathynella imuniensis Camacho, 1987 as outgroup representative of the Bathynellidae's sister lineage of Parabathynellidae Noodt, 1965 to root the phylogenetic analysis.All sequences used were submitted to GenBank (see Table 1 for locality, collection voucher number and GenBank Accession Number for each specimen).
Sequences obtained were then compared with sequences from GenBank (mostly generated by the authors of this paper over many years of wok) using Blast (Altschul et al. 1997).
Phylogenetic analyses were carried out using Maximum Likelihood (ML) and Bayesian Inference (BI) approaches, analyzing both the COI and 18S datasets separately and together.The ML analyses were conducted in IQ-TREE ver.2.1.1 (Minh et al. 2020).We estimated the best partition scheme with the option MFP-MERGE in ModelFinder (Kalyaanamoorthy et al. 2017), with the following parameters: low perturbation strength (-pers 0.2), number of unsuccessful iterations to stop (-nstop) set to 500 and, to assess node support, 2000 ultrafast bootstrap replicates.The BI analyses were run in MsBayes ver.3.2.6 (Ronquist et al. 2012) as implemented in CIPRES Science Gateway ver. 3 (Miller et al. 2010).The substitution model space was explored with the reversible-jump model (option lset nst = mixed rates = invgamma; Huelsenbeck et al. 2004).Two independent analyses were run with one cold and three heated chains, each chain ran for 100 million generations, with the first 25% discarded as burnin.From the resulting trees, a 50% majority rule consensus tree was obtained.The consensus phylogenetic tree was then edited in FigTree ver.1.4.3.(http://tree.bio.ed.ac.uk/software/figtree).

Description
MeasureMents and appearance.Body total length of holotype 1.12 mm.Total length of females 1.0-1.21mm.Body elongated, articles widening slightly towards posterior end, approximately ten times as long as wide.Head longer than wide.Pleotelson with one small barbed dorsal seta on each side.antennules (aI) (Fig. 2A).Seven-segmented; first three articles almost as long as last four articles combined; first article bit longer than the last article, which is more slender than the other articles; fourth article very short, fifth and sixth equal in length; inner flagellum almost square; setation as in Fig. 2A article six with three aesthetascs, similar in size; seventh article with three aesthetascs similar in size.AI slightly shorter than AII.antennae (AII) (Fig. 2B).Seven-segmented; first article similar in length to the fourth; second and third articles are the shortest, while the fifth is slightly longer and just over the half length of the distal article setal formula: 0+0/1+0/2+1/2+0/0+0/2+2/5. labruM (Fig. 2C).Almost trapezoidal; with smooth free edge and a median cleft.paragnaths (Fig. 2D).Almost rectangular, globose, with a very strong claw on distal part; dense setulation on distal half.
MandIbles (Md) (Fig. 2E-F).Palp with three articles, third article (Fig. 2E) with two strong barbed claws, first and third article almost square, second article elongated.Masticatory part (Fig. 2E): incisor process (pars incisiva) with two teeth; processus incisivus accessorius with one tooth and one small setalike tooth; pars molaris with one tooth, nearest to processus incisivus accessorius, bidentate, and with two dentate structures, parallel to main axis of teeth, each with two small denticles and with a strong distal tooth.
MaxIllules (MxI) (Fig. 2G).Proximal endite with four setae, three of them setulose; distal endite with six teeth (four with denticles and two seta-like); three plumose setae of different lenght, one longer than the other two, in outer margin.
pleopods (Fig. 4D).Two segmented; first article with very long plumose seta; second article with five setae of different length.
pleotelson (Fig. 4E).With one long, plumose dorsal seta at each side near base of furca.Furcal raMI (Fig. 4E).Almost square, bearing five short spines of similar size except second, which is slightly longer than rest and the dorsal, which is slightly shorter.
uropods (Fig. 4F).Sympod, almost square, as long as endopod, with five long, equal distal spines; endopod almost 20% longer than exopod, with three strong claws (basal two subequal in length, distal claw twice as long as the others), three very long distal barbed setae (Fig. 4I) and one plumose setae located dorsolaterally; exopod with four setae (two terminal and two medial).

Remarks
The new species shares the combination of morphological characters listed in the diagnosis with the type species of the genus Hobbsinella, described originally from Texas (Fig. 1B): seven-segmented AI and AII; pars molaris of mandible with two parts; endopod of all thoracopods four-segmented; female thoracopod VIII biramous and a very large epipod.The genus presents some peculiarities with respect to other North American and European genera, as already highlighted when describing the genus from Texas (Camacho et al. 2018b).The new species maintains those peculiarities of the type species but it is worth clarifying some of them.For example the fourth and last articles of AII in the species of Hobbsinella are longer than in other species of the American and European genera.The arrangement of the teeth on the pars molaris of the mandible is also unique.The female ThVIII has a very long epipod that exceeds the length of the basipod, but it is not bulky as in other genera with long epipods.The pleopod consists of two articles, as in all species of the family, but in most species, the first article is generally short (less than half the length of the second).In Hobbsinella, the first article of the pleopod is two thirds the length of the second.The spines of the sympod of the uropod are fairly long and not very thick, unlike most genera with shorter, thicker sympodal spines.The morphological differences between the species are difficult to find.The new species is slightly smaller than the type species, H. edwardensis.Hobbsinella gunnisonensis sp.nov.AII is slightly longer than AI (as Vandelibathynella Serban, Coineau & Delamare Deboutteville, 1971) while in H. edwardensis the great length of the AII is very striking, which far exceeds the length of AI (Table 3) (Camacho et al. 2018b).The third article of AI has only three setae on the new species, five in H. edwardensis, and also three aesthetacs on article six (only two in the type species), but the setal formula of the rest of articles is similar in both species and both species lacks of medial seta on exopod of AII.Both species differs also in the combinations of setae on the articles of the endopod of ThI to V, as well as the combinations of setae on the basipod of all thoracopods (Table 3).The differences between the two species are subtle and very difficult to appreciate, since in general they refer to the size, appearance and relative proportions of the different articles.The number of spines and/or setae on MxII, Ths and uropods differ between the two species, and these differences are summarized (Table 3) to facilitate comparison.

Molecular results
18S rRNA and COI sequences were obtained from five females specimens of the new species (Table 2).
The concatenated COI-18S data set is represented by 31 sequences of 1580 bp.509 bp COI and 1071 bp 18S sequences were obtained from 31 specimens.
One of these correspond to the subfamily Bathynellinae Grobben, 1905 and the other to the subfamily Gallobathynellinae Serban, Coineau & Delamare Deboutteville, 1971 in which the new species is placed.The subfamily Gallobathynellinae comprises species of the genera Vejdovskybathynella (PP = 0.9, BS = 82) and Paradoxiclamousella (PP = 1, BS = 100) from the Iberian Peninsula, Gallobathynella from France and Hobbsinella (PP = 1, BS = 100) from the USA with two well differentiated and supported clades (PP = 1, BS = 100

Discussion and conclusion
In groups such as bathyllenaceans, where morphological simplification is extreme, the validity of new species based exclusively on morphology may be questionable, specially in the absence of specimens of both sexes to complete comparisons.The basal Australian clade is formed by four genera, Pilbaranella, Muccanella, Fortescuenella and Anguillanella and corroborates previous results (Camacho et al. 2018a(Camacho et al. , 2018b(Camacho et al. , 2020(Camacho et al. , 2021;;Perina et al. 2019aPerina et al. , 2019b)).The taxonomic position of the new species seems clear based on our detailed morphological and molecular analysis.It would be interesting to obtain molecular information of the American species assigned to the genus Bathynella, to genetically compare them with the European species of Bathynella and see if they form a monophyletic clade.According to Serban (2000), it is unlikely that all species currently assigned to the genus Bathynella are really Bathynella.Researchers are identifying new morphological characters to further describe the male ThVIII, finding differences that morphologically justify the creation of new genera (e.g.Future studies of species within the family should incorporate DNA sequences, because as we are seeing with the most recent papers (Camacho et al. 2018a(Camacho et al. , 2018b(Camacho et al. , 2020(Camacho et al. , 2021(Camacho et al. , 2022;;Perina et al. 2018Perina et al. , 2019aPerina et al. , 2019bPerina et al. , 2022)), molecular information can help to resolve phylogeny and relationships amongst species and genera.Diversification within the family appears to be greater than previously thought.In a complex group with morphological homogeneity, such as Bathynellidae, where a few and difficult characters separate species and genera, the use of additional tools, such as molecular data, is fundamental to understand the diversity and the evolutionary patterns in different countries.

Distribution and paleobiogeography
Until the year 2000 only four species of the family Bathynellidae were known in North America, in two genera, Bathynella (B.riparia Pennak & Ward,1985, Fig. 1A;B. fraterna Cho &Kim, 1997 andB. germanitas Cho &Kim, 1997) and Pacificabathynella (P.sequoiae Schminke & Noodt, 1988) from Colorado and California.Three new species described in 2009 from Montana and one from Alaska (Camacho et al. 2009(Camacho et al. , 2016) ) expanded the distribution range for this family 3000 km further north.
In 2018, a new genus from Texas was described (Camacho et al. 2018b).With the description of H. gunnisonensis sp.nov.collected from Colorado, the distributional range of the genus is extended more than 1300 km.The two species of Hobbsinella also occur in very different drainage basins on opposite sides of the continental divide (Fig. 1A-B).
Numerous North American species have been collected but not formally described: Noodt (1974) reported bathynellids from California and Pennak & Ward (1985) reported bathynellids in the states of Montana, Wyoming, Colorado, Kansas, Oklahoma, Indiana, Ohio and Georgia.These reports show that there is a lot of work to be done on the North American Bathynellidae, and that the diversity described so far is only the 'tip on the iceberg'.It would be very interesting to discover whether the new collections of Bathynellacea were preserved in 100% etanol and refrigerated to be able to obtain DNA sequences even years after their collection and thus would be really useful in future studies.
Within Gallobathynellinae the genera Vejdovskybathynella, Paradoxiclamousella, Gallobathynella, and Hobbsinella are distributed in Europa (Spain, France, Germany, Switzerland and Italy) and North America (Texas and Colorado) (Camacho 2019).This subfamily is distributed across two continents with presumed Laurentian origins.Continental drift subdivided the area in the Early Triassic (245 to 205 Ma; Golonka 2007), when the separation of the Iberian Peninsula from North America started (Yilmaz et al. 1996).The genera Antrobathynella and Altainella, are distributed in Eurasia together with some undescribed species of Bathynella from Italy and Slovenia.Unfortunately, many species morphologically identified as Bathynella from different continents do not have sequenced data to support their position in the phylogeny, therefore the distribution of this genus is uncertain.
The study of the palaeobiogeography of Bathynellidae is difficult, due to their very ancient origins and lack of surface relatives (Coineau & Camacho 2013) and because of their morphological homogeneity.There are no fossils or surface bathynellids discovered so far, so their present-day ranges are influenced by a combination of more or less restricted habitats, lifestyles, and biogeographical patterns reflecting ancient hydrology.Plate tectonics appears to be the major vicariant process that influenced their evolutionary history at a world-wide scale (Coineau & Camacho 2013).The new species from Colorado described here belongs to a genus described on the other side of the continental divide (Fig. 1A-B) (Texas), and DNA confirmed the sister relationship between the two species.So the actual distribution means that they probably have a common ancestor that was widespread in the past, perhaps with Pangean or Laurentian distributions.

Fig. 1 .
Fig. 1.Field study area, showing the type locality (Lottis Creek, cross in circle).A. State of Colorado with known bathynellaceans Hobbsinella gunnisonensis Camacho & Taylor sp.nov.(white circles, cross in circle for type locality) and Bathynella riparia Pennak & Ward, 1985 (white triangle).Small black circles: 48 streambed sites sampled during the summer 2018 using a Bou-Rouch pump where Bathynellacea Chappuis, 1915 were not detected.Light blue shaded areas: approximate Late Pleistocene glacial extent from Leonard (2007: fig.2).Pink-brown shading: the Gunnison River Drainage Basin.Green-Southern Rockies US EPA Level 3 Ecoregion.Stream order (3-7) indicated by dark blue line width, 1 st and 2 nd order streams not shown.Red line: continental divide.Colorado counties shown as thin black lines.B. Conterminous United States in North America, with distributions of Hobbsinella edwardensis Camacho et al., 2018 (squares) and Hobbsinella gunnisonensis (circles).Colorado River Drainage Basin indicated in darker brown.
amplification, and sequencing DNA extraction and amplification methods have been described inCamacho et al.

Fig. 5 .
Fig. 5. Phylogenetic relationships among the species of the family Bathynellidae Grobben, 1904 included in this study.The Bayesian phylogenetic tree based on COI and 18S.Hobbsinella gunnisonensis Camacho & Taylor sp.nov. is highlighted in red.The same topology was recovered under a Maximum Likelihood approach.Support for each node is represented by the posterior probabilities (PP) resulting from the Bayesian Inference analysis and the bootstrap support values (BS) obtained for the Maximum Likelihood tree (PP/BS).C = cave.
EtymologyThe species name, gunnisonensis (adjective, patronym), is derived from the Gunnison River Drainage Basin in the headwaters of the Colorado River where the new species occurs in two tributaries of the Gunnison River.
).One clade includes H. edwardensis from Texas and the other includes the specimens of H. gunnisonensis sp.nov.from the two localities in Colorado.The subfamily Bathynellinae includes species of Bathynella from Slovenia and Italy; Antrobathynella stammeri (Jakobi, 1954) from the UK and Altainella calcarata Camacho et al., 2020 from Russia.The four species described from Western Australia (Anguillanella callawaensis Perina & Camacho, 2019, Fortescuenella serenitatis Perina & Camacho, 2019a, Muccanella cundelinensis Perina & Camacho, 2019 and Pilbaranella ethelensis Perina & Camacho, 2018) could represent Austrobathynellinae Delamare Deboutteville & Serban, 1973, but molecular data of the original species for which the subfamily was created are needed to confirm this.
(Abrams et al. 20121)sent all the morphological characters needed to establish the new species, although based only on females, because the molecular information obtained supports the decision to create a new species, H. gunnisonensis sp.nov., genetically distant from H. edwardensis.The molecular divergence (COI uncorrected p-distance) between the two species of Hobbsinella is 8.5-8.7%, which is lower than COI p-distance found amongst some European species of the genus Vejdovskybathynella (between 12-14%)(Camacho et al. 2011), but is higher than, for example, that found between species of the genus Brevisomabathynella Cho, Park & Ranga Reddy, 2006 (about 6%)(Abrams et al. 2012) of the family Parabathynellidae, therefore molecular data support the morphology and the decisión to erect a new species from only females.The use of COI thresholds is helpful in identifying potential new species, but their description should be based on multiple lines of evidence integrating molecular and morphological data.The molecular data provide the phylogenetic position of H. gunnisonensis sp.nov.as a sister species of H. edwardensis.The genus Hobbsinella is confirmed as sister group of the European genera of the subfamily Gallobathynellinae, well differentiated from the subfamily Bathynellinae, the other clade which includes Bathynella, Antrobathynella and Altainella.There are three major monophyletic and wellsupported groups: Gallobathynellinae and Bathynellinae subfamilies, and the Australian bathynellids.