Revalidation of the Spanish stone loach Barbatula hispanica (Lelek, 1987) (Teleostei, Nemacheilidae) according to morphological and mitochondrial data

– This study revalidates Barbatula hispanica, previously considered a junior synonym of B. quignardi . This species is found in the Ebro drainage and in Cantabria (Spain) as well as in the Adour drainage (Southwestern France). It is characterized by an upper lip with a well-marked medial incision and an interorbital width 18.5 – 33.7% of the HL. The species delineation is corroborated by the cytochrome oxidase subunit 1 molecular marker. We provide the sequence of 12S rDNA (950 bp) as reference for environmental DNA studies, and discuss also the taxonomy of B. quignardi which would be restricted to the Lez River.


Introduction
The stone loaches Barbatula spp (Teleostei, Nemacheilidae) are small freshwater teleost fishes occurring in brooks of most watersheds of Eurasia . They are rheophilic and live in high current environments, hiding under stones and vegetation (Kottelat and Freyhof, 2007;Keith et al., 2020).
The taxonomy of the group has long been discussed. At the end of the 20th century, Barbatula barbatula (Linnaeus, 1758) was considered widespread in Europe (Kottelat, 1997). However, molecular data have delineated a multitude of evolutionary lineages in the different populations of what was previously called B. barbatula. Šedivá et al. (2008) showed the paraphyly of B. barbatula with the cytochrome b (cytb) marker, due to the position of B. vardarensis (Karaman, 1928), B. sturanyi (Steindachner, 1892) and B. zetensis (Sorić, 2000).
They delineated at least five distinct evolutionary lineages in the Danube river basin, three lineages in the North Sea basin (corresponding to the drainages of the Elbe, the Dniester, and a Vistula þ Oder þ Salgir group), and three Mediterranean lineages corresponding to the Ebro, the Rhône and the Ter catchments. Later, with the development of DNA barcoding projects sensu Hebert et al. (2003) using the cytochrome oxidase subunit 1 (COI) marker, other evolutionary lineages were noticed. Mendel et al. (2012) delineated two evolutionary lineages in Czech Republic corresponding to the Elbe and Oder drainages. Knebelsberger et al. (2015) distinguished two other lineages corresponding to the Rhine and the upper Danube catchments, different from the Elbe and middle Danube drainages (Behrmann-Godel et al., 2017). Finally, Norén et al. (2018) analysed all 1154 sequences of the FREDIE project (www.fredie.eu) and found 13 other lineages, mostly in Russia, Ukraine and western Europe. Thus, according to mitochondrial data (cytb and COI), the genetic pattern resembles the one of the European minnows Phoxinus spp (e.g. Denys et al., 2020;Palandačić et al., 2020), with almost all European catchments having at least one endemic stone loach species.
Originally, Linnaeus (1758) described Cobitis barbatula following the description made by Artedi (1738) and Linnaeus (1746). The syntype specimens came from both Germany and Sweden. Therefore, it is unclear which evolutionary lineage the syntype specimens belong to, as there are four distinct lineages in Germany and stone loaches were introduced in Sweden from Germany and eastern European countries (see Norén et al., 2018). All the European stone loaches populations must therefore be considered as Barbatula spp.
However, several other European Barbatula species were described, and some are still valid, like Barbatula quignardi (Băcescu-Meşter, 1967 -Meşter, 1967. This Spanish form was described based on specimens from the Ibaizabal river at Durango (Nervión drainage) and the Tajo river. It can be characterized by a more cylindrical body, a shorter head, a larger eye diameter, rather long barbels and a rather narrow and long caudal peduncle (Băcescu-Meşter, 1967). Lelek (1987) made this infraspecific name available, calling it Noemaicheilus barbatulus hispanica Lelek, 1987. In 2007, Kottelat and Freyhof (2007 revalidated Barbatula quignardi (Băcescu-Meşter, 1967) based solely on morphological criteria (length of caudal peduncle 1.1 to 1.6 times in its depth (vs. 1.4 to 2.2 for B. barbatula). They extended its distribution from the Lez river to the South-West of France (Garonne, Adour and Mediterranean basins) and North-eastern Spain. And Kottelat (2012) designated Noemacheilus barbatulus forma hispanica Băcescu-Meşter, 1967 and Nemaicheilus barbatulus hispanica Lelek, 1987 as junior synonym of Barbatula quignardi.
However, according to Băcescu-Meşter (1967), Spanish populations differ from B. quignardi by a more vigorous size, a shorter head, a larger eye diameter, rather long barbels, a body more cylindrical, a caudal peduncle rather narrow and long, and a colouration with brown-black variegations reaching to lower half of body. This difference had however not been checked with molecular data. Combining morphological and molecular data led to the description of Barbatula leoparda Gauliard, Dettai, Persat, Keith and Denys, 2019 in French Catalonia, and highlighted the need for a revision of B. quignardi (Gauliard et al., 2019).
Here we provide the analysis of two widely used mitochondrial markers, in order to verify if Spanish populations are included in B. quignardi or if they should be considered a distinct species. The COI is the reference marker for DNA barcoding and 12S rDNA is used for environmental DNA (Valentini et al., 2016;Miya et al., 2020).

Sampling
Samples were collected by electrofishing between 2009 and 2016 from 21 locations (Fig. 1). After anaesthesia, Spanish fish were fixed in formalin 5% but fin-clips were conserved in 95% EtOH. As prescribed by the French legislation (substitution of formalin, article R. 4412-66), fish from France were fixed and preserved in 95% EtOH by using progressive concentration of EtOH over a few hours in order to lower the body shrivelling induced by osmotic shock.

Morphological analysis
Counts and measurements were taken from the left side following Kottelat and Freyhof (2007). Measurements were taken using an electronic caliper. All measurements were made point to point, never by projection. The two last dorsal and anal fin rays were counted as one because they are connected by the same pterygiophore. External characters link to the nostril positions and the lips following Prokofiev (2010Prokofiev ( , 2015 were observed.

Molecular delineation
ABGD and BIN analyses as well as the phylogenetic tree for the COI marker (609 bp) inferred from 55 sequences of Barbatula discriminate 3 clades within the ingroup (Fig. 2). The first clade is a single haplotype (three sequences) of B. quignardi from its type locality (Lez River). Its sister group is composed by one clade with the two specimens (2 haplotypes) of B. leoparda, and a second clade with specimens from the Adour drainage (Southwestern France), Ebro drainage and Basque coastal catchments (Artibai, Butrón, Nive, Nivelle, Nervión and Oka; Northeastern Spain). This last clade is separated from B. quignardi and B. leoparda by an average divergence of respectively 1.6 and 2.1%. Within this clade, the seven haplotypes have very little geographical structuration and an average divergence of 0.1%. The COI haplotype network is congruent with the clades recovered in the phylogenetic analysis (Fig. 3). It highlights that samples from the Adour, Nive, Nivelle and Baias catchments each have their own haplotypes.

Diagnosis
Barbatula hispanica is distinguished from B. leoparda by an upper lip with a well-marked medial incision (vs. with a medial incision not exceeding the half of its width; Fig. 5), a fleshy upper lip (vs. slim; Fig. 5), well-marked mental lobes on the lower lip (vs. not well marked; Fig. 5), an interorbital width 18.5-33.7% HL (vs. 35.5-41.8% HL; Tab. 1; Fig. 6), a post-   orbital length representing 36.9-48.2% of HL (vs. 45.7-51.6% HL; Tab. 1), the third pair of barbels at the corner of the lips reaching the posterior eye border (vs. not reaching to the posterior eye border; Fig. 7), and a belly and jugular area without blotches (vs. presence of blotches; Fig. 8).

Distribution and habitat
Barbatula hispanica occurs in the Ebro and in the eastern Cantabria coastal catchments in Spain, as well as in the Adour drainage (Southwestern France) (Fig. 1). Like other Barbatula species, it lives in fresh, clear waters with current and grounds of stones, pebbles, gravels, sand or marl (Neveu, 1981;Mastrorillo et al., 1996;Doadrio et al., 2011).

Nomenclatural note
Băcescu-Meşter (1967) described Noemacheilus barbatulus forma hispanica from eight specimens from the river Ibaizabal at Durango (Nervión drainage) and three specimens from the Tajo River. However, no catalog number is mentioned in the article. There is only a figure illustrating the taxon with a male (length 8.6 cm TL) caught in the Nervión drainage in April 1961(Băcescu-Meşter, 1967: Fig. 6A-B). There are two lots of Barbatula from the Nervión drainage (river Ibaizabal) at Durango in the MGAB collections (MGAB400.141 and MGAB400.151) and both were caught in October 1949 but we have no certainty on whether they belong to the syntype series (Iftime, pers. com.). Moreover, according to Doadrio et al. (2011), no loach inhabits the Tajo River. So we do not know for sure the origin of the three syntypes from this locality, and they may even correspond to another evolutionary lineage or species. By consequence, according to the art. 74.7 of the International Code of Zoological Nomenclature, we designate as lectotype of Noemacheilus barbatulus forma hispanica Băcescu-Meşter, 1967 the specimen illustrated by Băcescu-Meşter (1967: Fig. 6A-B). And then, we designate this same specimen as lectotype of Nemacheilus barbatulus hispanica Lelek, 1987.

Discussion
Both molecular and morphological data agree on the delineation of Barbatula hispanica. It therefore needs to be revalidated. This species occurs in Spain in the Ebro drainage and in the eastern Cantabria coastal catchments as well as in the Adour drainage (Southwestern France). This distribution is identical to the one of two other species with which it co-occurs, Gobio lozanoi Doadrio and Madeira, 2004 and Phoxinus bigerri Kottelat, 2007 (Doadrio andMadeira, 2004;Kottelat and Persat, 2005;Kottelat, 2007;Doadrio et al., 2011;Corral-Lou et al., 2019;Denys et al., 2020). These three species would share the same biogeographical history in the Iberian Peninsula, with the Pleistocene glaciation cycles and the apparition of Mediterranean climate (see Corral-Lou et al., 2019), as well as a possible connection between southern France and northern Spain during the Pleistocene when water levels decreased due to glaciation cycles (Patarnello et al., 2007). The low intraspecific genetic distance (0.1%) in addition to the low geographical structuration observed for the COI marker may suggest a recent colonization of some catchments, like the Ebro drainage, due to the glaciers melting during the Late Pleistocene in the Cantabrian belt (Serrano et al., 2013(Serrano et al., , 2017García-Ruiz et al., 2016). Local introductions like in northern Europe (Lundberg and Svanberg, 2010;Norén et al., 2018) or like in the Neretva catchment in Croatia (Tutman et al., 2017) cannot be excluded.
Barbatula hispanica can be distinguished from both other species with the COI marker (Fig. 2). The genetic distance from B. leoparda (>2%) is usual for teleosts (e.g. Ward et al., 2009;Dettai et al., 2011;Geiger et al., 2014). The genetic distance with B. quignardi is lower (1.6%) but in line with the interspecific distances observed for 11.3% of Mediterranean freshwater fishes (Geiger et al., 2014). Both ABGD and BOLD detected barcode gaps between these species, highlighting distinct evolutionary lineages.
Barbatula hispanica is also distinguished from both other species by morphological characters. Reevaluating the diagnosis of Băcescu-Meşter (1967), we disagree with the use of the head, the caudal peduncle, the barbels lengths and the eye diameter to distinguish B. hispanica from B. quignardi.
The integration of more specimens in our study made overlaps apparent between these characters.
Barbatula quignardi seems to be restricted to the Lez River, contrary to what Kottelat and Freyhof (2007) thought. It differs from B. hispanica by an upper lip with a medial incision not exceeding the half of its width (vs. with a well-marked medial incision; Fig. 5) and an interorbital width −29.3-36.2% HL (vs. 18.5-33.7% HL; Tab. 1; Fig. 6). It also differs from B. leoparda by an upper lip with a well-marked medial incision (vs. with a medial incision not exceeding the half of its width; Fig. 5), a fleshy upper lip (vs. slim; Fig. 5), well-marked mental lobes on the lower lip (vs. not well marked; Fig. 5), an interorbital width 29.3-36.2% HL (vs. 35.5-41.8% HL; Tab. 1; Fig. 6), a post-orbital length representing 40.0-45.8% of HL (vs. 45.7-51.6% HL; Tab. 1), the third pair of barbels at the corner of the lips reaching the posterior eye border (vs. not reaching to the posterior eye border) and a belly and jugular area without blotches (vs. presence of blotches). Geiger et al. (2014) grouped samples from the Rhône river with this species according to mitochondrial data. However, Băcescu-Meşter (1967) and Gauliard et al. (2019) highlighted morphological differences between the populations from the Lez River and the Rhône catchment. We prefer to consider each population separated until more data is available. This situation is similar to the one of the Lez sculpin, Cottus petiti Bǎcescu and Bǎcescu-Meşter, 1964, endemic to the Lez River. It is close to Cottus gobio Linnaeus, 1758 occurring in the Rhône catchment, different according to morphological data (Freyhof et al., 2005) but not distinguishable with molecular data (Eppe et al., 1999;Šlechtová et al., 2004;Geiger et al., 2014).
This study furthers the evolving taxonomic knowledge on Barbatula in Europe, with several species described or revalidated (Sorić, 2000;Geiger et al., 2014;Gauliard et al., 2019) and multiple cryptic lineages highlighted (Šedivá et al., 2008;Knebelsberger et al., 2015;Behrmann-Godel et al., 2017;Norén et al., 2018;Behrens-Chapuis et al., 2021). Barbatula hispanica is the fourth stone loach species known in France, and the second species known for Spain after B. cf barbatula. However, the population in the Ter catchment (Northeastern Spain) belongs to a distinct evolutionary lineage from the Ebro populations and those of other European catchments (Šedivá et al., 2008), and they are also morphologically different (Aparicio et al., 2016). Moreover, a population introduced in the Duero drainage is also morphologically different . These populations were originally affiliated to B. barbatula , but without molecular data. They could actually correspond to any Barbatula sp.