A widespread Ponto-Caspian invader with a mistaken identity: integrative taxonomy elucidates the confusing taxonomy of Trichogammarus trichiatus (= Echinogammarus) (Crustacea: Amphipoda)

Abstract

Amphipods have diversified greatly in the Ponto-Caspian region. Although many of these species are prominent invaders their systematics remains unclear. Taking an integrative approach, we investigate the taxonomy of Trichogammarus trichiatus, a widespread invader in European inland waters. It was initially described from the north-eastern Black Sea coast as Chaetogammarus trichiatus by Martynov in 1932. A similar taxon, Chaetogammarus tenellus major, was described by Cărăușu from the western Black Sea in 1943 but later synonymized with C. trichiatus. Chaetogammarus trichiatus was itself shuffled between Chaetogammarus and the Atlanto-Mediterranean Echinogammarus, currently being assigned to Trichogammarus. Our analyses (six DNA markers, 60 measurements and scanning electron microscopic imaging) reveal that T. trichiatus and C. tenellus major are distinct species; the former is a Caucasian endemic, whereas the latter invaded Europe. Unexpectedly, T. trichiatus is an incipient species molecularly nested in Chaetogammarus ischnus, despite pronounced morphological and geographical differentiation. We also recover Chaetogammarus as polyphyletic, yet its member species are nested in the Ponto-Caspian radiation, thus distinct from Echinogammarus. Consequently, we reassign T. trichiatus to Chaetogammarus (Chaetogammarus trichiatus), synonymize Trichogammarus with Chaetogammarus and place C. tenellus major in the new genus Spirogammarus gen. nov. (Spirogammarus major comb. & stat. nov.). Chaetogammarus necessitates further systematic refinement.

INTRODUCTION

The Ponto-Caspian region comprises the Aral, Azov, Black and Caspian Seas, in addition to the lower water courses of their catchments. These waterbodies are remnants of the once widespread epicontinental Paratethys Sea that stretched from the Alps to the Himalayas (Popov et al., 2004; Palcu et al., 2021). The area is an evolutionary hotspot, characterized by endemic aquatic faunas that have broad tolerance to fluctuations in salinity (Reid & Orlova, 2002; Krijgsman et al., 2019). Gobiid fishes, molluscs and especially crustaceans have undergone extensive radiations in this region throughout the last 15 Myr (Mordukhai-Boltovskoi, 1979; Audzijonyte et al., 2008; Naseka & Bogutskaya, 2009; Neilson & Stepien, 2009; Sands et al., 2019; Wesselingh et al., 2019; Copilaș-Ciocianu & Sidorov, 2022).

Amphipod crustaceans are one of the most diverse groups of Ponto-Caspian taxa, comprising ≥ 96 known endemic species (Pjatakova & Tarasov, 1996; Copilaș-Ciocianu & Sidorov, 2022). Of these, 82 belong to a large, apparently monophyletic clade, termed the Ponto-Caspian gammaroid radiation because it comprises several families that belong to the superfamily Gammaroidea (Copilaș-Ciocianu & Sidorov, 2022). This radiation exhibits a remarkable diversity in terms of morphology and ecology, on a par with the iconic Lake Baikal amphipod radiations. Ponto-Caspian amphipods can be found from mountain springs to depths > 500 m, on basically every type of substrate, including symbionts. Morphologically, they display a great diversity, ranging from minute (< 5 mm) symbiotic species with attenuated appendages to large (> 2 cm) species characterized by great diversity in body shape, appendage length and ornamentation (Copilaș-Ciocianu & Sidorov, 2022).

The evolutionary success of these amphipods is likely to be attributable to their high adaptability and environmental tolerance (Reid & Orlova, 2002; Rewicz et al., 2014; Borza et al., 2017; Cuthbert et al., 2020; Copilaș-Ciocianu & Sidorov, 2022). Consequently, nearly 40% of Ponto-Caspian amphipods have dispersed beyond their native range and are considered alien or invasive (Copilaș-Ciocianu et al., 2023). Their alien ranges are constantly expanding (Lipinskaya et al., 2021; Copilaș-Ciocianu & Šidagytė-Copilas, 2022). Perhaps in all cases, this dispersal is related to anthropogenic changes, such as the increased ion content of inland waters, shipping, intentional introductions and canals that connect formerly isolated watersheds (Bij de Vaate et al., 2002; Grigorovich et al., 2002). The arrival of Ponto-Caspian amphipods is usually followed by restructuring of ecological communities and the local extinction of native amphipod species (Dermott et al., 1998; van Riel et al., 2006; Arbačiauskas, 2008; Grabowski et al., 2009). The superior competitive abilities of the Ponto-Caspian invaders are probably attributable to their broader environmental tolerance (Šidagytė & Arbačiauskas, 2016), increased fecundity (Grabowski et al., 2007), aggressiveness (Dick & Platvoet, 2000; Bacela-Spychalska & Van der Velde, 2013) and unpalatability to predators (Błońska et al., 2016).

Despite the negative effects of Ponto-Caspian invasive amphipods, the taxonomy of some species is shrouded in confusion, especially owing to incomplete species descriptions (Copilaș-Ciocianu & Sidorov, 2022). The recent increase in the utilization of molecular methods has shown that some invasive species comprise multiple evolutionarily independent lineages in their native range or belong to a single molecular lineage with morphologically distinct taxa (Jażdżewska et al., 2020; Copilaș-Ciocianu et al., 2022a; Morhun et al., 2022). These studies question the taxonomic validity of some previously described taxa, with important ramifications for conservation and biomonitoring.

A taxonomically problematic, yet widespread, invasive species in European inland waters is Trichogammarus trichiatus (Martynov, 1932), alternatively known as Chaetogammarus trichiatus or Echinogammarus trichiatus (Rachalewski et al., 2013; Copilaș-Ciocianu et al., 2023). It is native to lagoons and estuaries in the north-western Black Sea (Rewicz et al., 2016; Copilaș-Ciocianu et al., 2023), but was first reported as an alien in Germany in 1996 (Weinzierl et al., 1997). It has since spread both upstream and downstream in the Danube River (Borza, 2009; Boets et al., 2012), currently inhabiting its entire length, in addition to the Rhine and Oder drainages in Central Europe (Copilaș-Ciocianu et al., 2023). It has also been reported from the upper stretch of the Dnieper River in Belarus (Lipinskaya, Radulovici & Makaranka, 2018).

The taxonomic status of T. trichiatus is confusing. It was initially described as C. trichiatus by Martynov in 1932 from the Khosta River in Russa at the foothills of the south-western Caucasus Mountains, flowing into the north-eastern Black Sea (Martynov, 1932). The description was detailed but poorly illustrated. A decade later, Cărăușu (1943) described Chaetogammarus tenellus majorCărăușu, 1943 from the western Black Sea lagoons in Romania and Bulgaria. Although this description was accompanied by detailed drawings, some authors considered C. tenellus major as a junior synonym of C. trichiatus (Straškraba, 1969; Dedju, 1980; Jazdzewski & Konopacka, 1988), and this prevails today (Rachalewski et al., 2013). Hou & Sket (2016) placed C. trichiatus in the newly erected monotypic genus Trichogammarus, but the synonymy of C. tenellus major with T. trichiatus has never been clearly justified. Based on the original descriptions, the two taxa were clearly separated by crucial characters, such as the armature of the urosome (solitary spines in T. trichiatus vs. clusters of two to four spines in C. tenellus major), shape of anterior head lobes (triangular in T. trichiatus vs. straight in C. tenellus major), the length of appendages (significantly longer and more slender in T. trichiatus) and the setation of gnathopod propodi (straight in T. trichiatus vs. characteristically coiled in C. tenellus major). Moreover, recent DNA barcoding studies have shown that the two taxa belong to distinct mitochondrial lineages, but surprisingly revealed that T. trichiatus was closely related to Chaetogammarus ischnus (Stebbing, 1899) (Copilaș-Ciocianu et al., 2022a).

Given this conflicting evidence, the aim of the present paper is to resolve the taxonomy of T. trichiatus by taking an integrative taxonomic approach that combines fine-scale morphometry, scanning electron microscopy (SEM), multilocus DNA markers and geography. Additionally, we also aim to bring further phylogenetic insight into the Ponto-Caspian endemic genus Chaetogammarus by sequencing a set of DNA markers from all known species, with the aim of facilitating its future systematic revision.

MATERIAL AND METHODS

Sampling

Sampling was conducted between 2009 and 2021 and designed to cover the native and invasive ranges (where applicable) comprehensively (see Supporting Information, Table S1). Besides T. trichiatus and C. tenellus major, we also examined C. ischnus, because previous DNA barcoding data suggest that it is closely related to T. trichiatus (Copilaș-Ciocianu et al., 2022a). We obtained specimens from type localities or as close as possible, but failed to secure C. ischnus from the Caspian Sea drainage despite intense sampling efforts. This gap was mitigated by the availability of COI sequences from this area from previous studies (see Phylogeography section below). Animals were collected along the shoreline of various seas, lagoons, rivers and estuaries, with the help of a hand net. Specimens were fixed directly in 96% ethanol in the field. Subsequently, they were identified under a stereomicroscope using the latest keys (Copilaș-Ciocianu & Sidorov, 2022).

Laboratory protocols, sequencing and alignment

Processing of samples for sequencing was done at the Nature Research Centre (NRC), Vilnius, Lithuania, at the Canadian Center for DNA Barcoding (CCDB), Guelph, ON, Canada or at the Department of the Invertebrate Zoology and Hydrobiology (DIZH), University of Lodz, Lodz, Poland (Supporting Information, Table S1). Genomic DNA were extracted as follows: at NRC using the Quick-DNA Miniprep Plus Kit (Zymo Research; details provided by Copilaș-Ciocianu et al., 2022a), at CCDB with an automated protocol (https://ccdb.ca/site/wp-content/uploads/2016/09/CCDB_DNA_Extraction.pdf; details provided by Morhun et al., 2022) and at DIZH using a standard Chelex procedure (details provided by Csabai et al., 2020).

Six markers were targeted for polymerase chain reaction (PCR) amplification: two mitochondrial fragments [cytochrome c oxidase subunit I (COI) and the large ribosomal subunit RNA (16S)]; and four nuclear markers [large ribosomal subunit RNA (28S), glutamyl-prolyl-tRNA synthetase (EPRS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and histone H3 (H3)]. Details on primers, PCR conditions and amplification are presented in the Supporting Information (Table S2). Sequencing was performed at CCDB or commercially by BaseClear and Macrogen. All chromatograms were inspected for contamination using BLAST (Altschul et al., 1990). All the newly obtained sequences were submitted to GenBank (COI: ON257949, ON257967, ON257994, ON258021 and OP466419–OP466517; 16S: ON258183, ON258198, ON258236 and OP466369–OP466418; 28S: OP466518–OP466576 and OP620914; H3: OP696981–OP697047; EPRS: OP466643–OP466708 and OP620915; and GAPDH: OP466577–OP466588). The molecular datasets are also available in the BOLD repository (http://dx.doi.org/10.5883/DS-DCTRICH).

Protein-coding sequences (COI, EPRS, GAPDH and H3) were aligned using MUSCLE (Edgar, 2004) in MEGA v.6 (Tamura et al., 2013) and amino acid translated to check for pseudogenes (indicated by stop codons and/or reading frame shifts). The nuclear protein-coding EPRS, GAPDH and H3 chromatograms contained heterozygous sites where double peaks were present. These markers were assigned to their corresponding alleles using PHASE (Stephens et al., 2001) implemented in DnaSP v.6 (Rozas et al., 2017). The 16S and 28S sequences were aligned with MAFFT7 (Katoh & Standley, 2013) using the G-INS-i method.

Phylogenetic analyses

The phylogenetic analyses aimed to place the evolutionary relationships of the focal taxa in a broader context and to examine the monophyly of Chaetogammarus. As such, a dataset was assembled that contained 31 species representing the major known lineages of the Ponto-Caspian gammaroid radiation, including the five known species of Chaetogammarus. The outgroup consisted of three taxa belonging to Echinogammarus s.l., which are the nearest relatives of Ponto-Caspian gammaroids (Hou & Sket, 2016). Additionally, we included two Pontic species of Echinogammarus that have morphological affinities with Chaetogammarus. Given that they were previously sequenced only for COI (i.e. Echinogammarus karadagiensisGrintsov, 2009 and Echinogammarus mazestiensisMarin & Palatov, 2021), their phylogenetic affinities were not assessed critically (Grintsov, 2009; Marin & Palatov, 2021; Copilaș-Ciocianu et al., 2022a). Details regarding taxa and collection sites are presented in the Supporting Information (Table S1).

The alignment for the 36 species and five markers (COI, 16S, 28S, EPRS and H3) had a total length of 3032 bp. Marker concatenation was done with SequenceMatrix (Vaidya et al., 2011). The GAPDH marker was not included because it was amplified successfully in a only few species, including the focal ones. It was used only in the phylogeography and species delimitation analyses (see Molecular species delimitation section below).

Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI). Optimal substitution models and partitions were selected with PartitionFinder (Lanfear et al., 2012). Maximum likelihood trees were inferred with raxmlGUI v.2.0 (Edler et al., 2021) using the RAxML HPC-PTHREADS-SSE3 binary (Stamatakis, 2014) and with IQTREE on a dedicated webserver (http://iqtree.cibiv.univie.ac.at; Trifinopoulos et al., 2016). For RAxML, we conducted a thorough ML search under the GTR+Г model applied to each partition, followed by 1000 rapid bootstrap replications. The IQTREE tree was obtained using the GTR+I+Г model applied to each partition. Branch support was assessed using 1000 ultrafast bootstrap replicates and the Shimodaira–Hasegawa approximate likelihood ratio test (SHaLRT). Bayesian analysis was performed with MrBayes v.3.2.6 (Ronquist et al., 2012). Two independent runs, each consisting of four Markov chain Monte Carlo chains, were run for 5 million generations, with a thinning of 1000 and a burn-in proportion of 0.3. The temp parameter was set to 0.08. All parameters were unlinked, and rates were allowed to vary independently. Effective sample size and convergence were calculated in Tracer v.1.7 (Rambaut et al., 2018). Bayesian analyses were repeated two times to assess the consistency of the results.

Phylogeography

For phylogeographical analyses, we constructed a dataset containing the three focal taxa, T. trichiatus, C. tenellus major and C. ischnus. For COI, we combined our new data with all the available sequences from GenBank and the Barcode of Life Data Systems (BOLD; https://www.boldsystems.org/; Ratnasingham & Hebert, 2007) obtained from previous studies (Cristescu et al., 2004; Radulovici et al., 2009; Rewicz et al., 2016; Lipinskaya et al., 2018; Copilaș-Ciocianu et al., 2022a). See the Supporting Information (Table S1) for further details. The COI haplotypes were selected using the online tool FaBox v.1.61 (https://birc.au.dk/~palle/php/fabox/; Villesen, 2007). For a subset of individuals, we sequenced the 16S, 28S, EPRS, GAPDH and H3 markers. The specimens were chosen based on preliminary observations regarding COI divergence (distinct subclades or haplogroups) or geographical representativeness.

Haplotype networks were constructed separately for each marker in Haploviewer (Salzburger et al., 2011) using ML trees constructed with MEGA v.6. To visualize patterns of mitochondrial and nuclear divergence, we constructed neighbour-net networks in SplitsTree v.4 (Huson & Bryant, 2006) based on concatenated mitochondrial (COI and 16S) or nuclear (28S, EPRS, GAPDH and H3) marker alignments. For both the haplotype and neighbour-net networks, only the phased alleles of the nuclear markers were used, in order to extract as much phylogenetic information as possible.

In order to link the observed patterns of genetic differentiation with putative historical factors, we performed a molecular dating analysis in BEAST v.1.8.2 (Drummond et al., 2012). The dataset consisted of concatenated COI and 16S haplotypes. The analysis was run for 10 million generations with a thinning of 1000. Speciation was modelled using the Yule process. The COI marker was split into codon positions. Convergence and mixing were assessed with Tracer v.1.7 after discarding the first 10% of states as burn-in. Divergence times were estimated using a strict molecular clock applied to each marker, because we are dealing mainly with shallow intraspecific variation (Copilaș-Ciocianu et al., 2022a). Dating intraspecific divergence is often fraught with issues owing to the difficulty of finding proper calibrations for recent time scales (fossils or biogeographical). Furthermore, rates of molecular evolution seem to be time dependent, with intraspecific rates being an order of magnitude faster than interspecific ones (Audzijonyte & Väinölä, 2006; Ho et al., 2011). Taking these issues into account, we opted for a strategy that combines both intra- and interspecific substitution rates. As such, a uniform prior distribution was chosen for the COI substitution rate, with the lower bound set to the amphipod COI interspecific rate of 0.0177 substitutions/Myr (based on fossil calibration from the study by Copilaș-Ciocianu et al., 2019a) and the upper bound set to the relatively fast intraspecific rate of 0.0329 substitutions/Myr observed in the mantis shrimp Haptosquilla pulchella (based on expansion dating from the study by Crandall et al., 2012).

Molecular species delimitation

The aim of these analyses was to test the evolutionary distinctiveness of the focal taxa at the molecular level. We used both single- and multilocus methods. The single-locus methods were based on the mitochondrial markers, consisting of two datasets (depending on method requirements): a COI haplotype dataset and/or a concatenated COI + 16S haplotype dataset. The methods included both distance- and tree-based approaches, with or without a priori divergence thresholds.

The distance-based methods were:

1. the barcode index number (BIN) (Ratnasingham & Hebert, 2013), which uses a refined single-linkage algorithm implemented in BOLD, whereby the newly submitted COI sequences are compared pairwise with all sequences in this database.

2. a non-threshold method that identifies barcode gaps by using a hierarchical clustering algorithm to assemble species by automatic partitioning (ASAP) (Puillandre et al., 2021). The analysis was run on a dedicated webserver (https://bioinfo.mnhn.fr/abi/public/asap/), based on Kimura two-parameter (K2P) distances and default settings.

The single-locus tree-based methods were as follows:

1. A non-threshold method that uses an input phylogram to distinguish coalescent vs. speciation processes assuming a Poisson distribution, Poisson tree processes (PTP; Zhang et al., 2013). The analysis was run on a dedicated webserver (https://species.h-its.org/) for 500 000 generations, with thinning of 100 and 0.1 burn-in, where the input phylogram was the ML tree constructed with IQTREE.

2. A non-threshold method that determines the population- to species-level transition by using a generalized Yule model, the generalized mixed Yule coalescent (GMYC; Pons et al., 2006). The analysis was run with the iTaxoTools v.0.1 software (Vences et al., 2021), using as an input the BEAST chronogram mentioned above.

3. A patristic distance threshold, which assumes that crustacean lineages separated by ≥ 0.16 substitutions per site at the COI locus are likely to be different species, the patristic distance threshold (PDT; Lefébure et al., 2006). Patristic distances were calculated with Patristic v.1.0 (Fourment & Gibbs, 2006), using as input an ML tree calculated with IQTREE.

All the methods (distance-based and tree-based) above used the COI + 16S dataset, except for BIN and PDT, which were used only on COI because they were developed specifically for this marker.

Multilocus delimitation was performed using the Bayes factors species delimitation (BFD) method (Grummer et al., 2014). The method identifies which of the a priori chosen groups of individuals best explains the data at hand by using Bayes factors (Kass & Raftery, 1995). Our aim was to test whether T. trichiatus and C. ischnus are distinct species, considering the observed low genetic divergence (see Results). Moreover, given that C. ischnus contains several mitochondrial lineages that are as divergent as T. trichiatus (see Results), we also tested a model where C. ischnus comprises three additional species based on the BIN approach. Although the BIN method delimited a total of seven putative species within C. ischnus, only three could be studied, because the others lacked nuclear data. To implement the method, we used a Bayesian implementation of the multispecies coalescent in BEAST v.1.8.2 using the *BEAST package (Heled & Drummond, 2010). We tested the following three hypotheses: (1) T. trichiatus and C. ischnus are distinct species; (2) T. trichiatus and C. ischnus are conspecific; and (3) C. ischnus comprises cryptic species. The marginal log likelihood of each model was calculated using path sampling (Baele et al., 2012, 2013) with a chain length of 100 000, 20 path steps, and α set to the default 0.3. Bayes factors were calculated as twice the difference between the marginal likelihood of the best-ranking model vs. its competitor. A value higher than ten indicates strong evidence against the null hypothesis. The BFD analysis was run in three possible combinations: nuclear markers only, mitochondrial markers only, and both marker sets simultaneously (only phased nuclear markers were used). Each combination was run in triplicate to ensure convergence.

Morphology and scanning electron microscopy

For morphological analyses, we included all three focal taxa: T. trichiatus, C. tenellus major and C. ischnus. We measured ten individuals per sex for each taxon, totalling 60 individuals. Differentiation among taxa was assessed by measuring 60 morphological traits per individual (of which four were meristic counts). These traits were chosen to cover, as much as possible, the functional aspects of the animals and to reflect taxonomically informative differences (Supporting Information, Table S3). The landmarks and function of these traits were established in previous studies (Fišer et al., 2009; Hutchins et al., 2014; Kralj-Fišer et al., 2020; Copilaș-Ciocianu et al., 2021; Premate et al., 2021; Copilaș-Ciocianu & Sidorov, 2022).

Before dissection, the cuticle of the animals was softened and partly cleared by overnight immersion in a 2% lactic acid solution and subsequent transfer for 3-4 hours to a 1:1 mixture of 70% ethanol and glycerine. Animals were dissected under a Nikon SMZ1000 stereomicroscope using fine tweezers, needles and microsurgical scissors. The dissected appendages were mounted temporarily in glycerol on microscope slides and photographed using a Pixelink M15C-CYL digital camera attached to either a Nikon SMZ1000 stereomicroscope or a Nikon Si microscope. Morphometric measurements were made with Digimizer v.4 software (https://www.digimizer.com/). Given that some of the mouthparts are asymmetrical in gammarids, we used only right-sided appendages. For the rest of the animal, we used either right- or left-sided appendages depending on specimen completeness. Raw morphometric measurements are available in the Supporting Information (Table S3) and at Figshare (https://doi.org/10.6084/m9.figshare.21610182).

Before statistical analyses, all measurements were regressed against body length to correct for differences in body size among specimens and regression residuals were used in subsequent analyses. All meristic counts were ln-transformed before the regression. Differentiation among taxa was assessed using a permutational multivariate analysis of variance (PERMANOVA) test with 9999 permutations and a Euclidean similarity index, with sequential Bonferroni correction for multiple comparisons. Sexes were analysed separately owing to the strong sexual dimorphism known in gammarids. For a visual interpretation of differentiation in morphospace, a principal components analysis (PCA) was performed on a correlation matrix. To visualize patterns of morphological differentiation, a dendrogram was constructed with Ward’s method and a Euclidean distance matrix based on squared Mahalanobis distances computed from the first three PCA axes. All statistical analyses were performed in PAST v.4 (Hammer et al., 2001).

For SEM imaging, we used male individuals of T. trichiatus, C. tenellus major and C. ischnus.

Dissected appendages were dehydrated in an ethanol series, air dried and sputter coated with gold (11 nm). Images were produced with a PHENOM PRO X SEM at the DIZH.

RESULTS

Phylogeny

The ML and BI concatenated phylogenetic analyses produced trees with similar topologies. Differences were observed only at unsupported nodes (Fig. 1). The phylogenies reveal that Chaetogammarus is polyphyletic, with its component species deeply nested within the Ponto-Caspian gammaroid radiation. However, the exact relationship among most species remains unresolved. Only Chaetogammarus warpachowskyi (Sars, 1894) has a well-resolved position. Chaetogammarus pauxillus (G.O. Sars, 1896) might be the earliest-splitting member of the genus, though this is weakly supported. The remaining species [Chaetogammarus hyrcanusPjatakova, 1962, C. ischnus, Chaetogammarus placidus (G.O. Sars, 1896) and C. tenellus major] form a polytomy at the base of a strongly supported clade that contains all the fossorial taxa (‘Pontogammaridae’). All analyses clearly support the distinctiveness of Chaetogammarus species from the Atlanto-Mediterranean Echinogammarus s.l. The phylogenies also confirm that the two Pontic taxa previously assigned to Echinogammarus (i.e. E. karadagiensis and E. mazestiensis) belong to the Ponto-Caspian gammaroid radiation.

With respect to the focal taxa, the phylogeny reveals that T. trichiatus and C. tenellus major represent clearly distinct clades that are not closely related. However, T. trichiatus is recovered as nested within the widespread species C. ischnus (Fig. 1).

Phylogeography

The haplotype networks reveal that all three focal taxa possess unique COI and 16S haplotypes (Fig. 2A). In the case of COI, the haplotypes of T. trichiatus are nested among those of C. ischnus, whereas in the case of 16S they remain slightly more distinct. At both markers, C. tenellus major is distant from the T. trichiatus–C. ischnus group, differing by 84 (16S) and 126 (COI) substitutions. However, at the nuclear level, T. trichiatus and C. ischnus share a number of haplotypes at each sequenced marker. In the case of EPRS, one of ten haplotypes are shared, for 28S two of six, for GAPDH two of five, and for H3 two of three (Fig. 2A). At all nuclear markers, the haplotypes of C. tenellus major are clearly distinct from the T. trichiatus–C. ischnus group, showing from five (H3) to 33 substitutions (28S) (Fig. 2A).

The mitochondrial neighbour-net networks indicate that T. trichiatus haplotypes group into two branches, which are situated among the branches of C. ischnus. Chaetogammarus tenellus major is situated at a much greater distance (Fig. 2B). At the nuclear level, T. trichiatus and C. ischnus are recovered as somewhat distinct, albeit close. Again, C. tenellus major is situated at a much greater distance (Fig. 2B).

The time-calibrated tree also confirms that T. trichiatus is nested in C. ischnus, although it forms a distinct sublineage that diverged ~0.41 Mya [95% highest posterior density (HPD): 0.22–0.65 Mya; Fig. 3]. The coalescent time for the T. trichiatus–C. ischnus clade is dated at 1.32 Mya (95% HPD: 0.83–2.00 Mya; Fig. 3). At this time, the earliest split occurred, when the Caspian lineage of C. ischnus diverged from the Black Sea lineages. The coalescent time for C. tenellus major is dated at ~0.2 Mya (95% HPD: 0.10–0.34 Mya). The T. trichiatus–C. ischnus clade and C. tenellus major split > 7.5 Mya (95% HPD: 5.5–12.3 Mya; Fig. 3). However, this divergence is most likely to be underestimated owing to the use of a strict clock.

The geographical distribution of the focal taxa (Fig. 4) shows that C. tenellus major is widespread in lagoons and estuaries along the north-western shore of the Black Sea. Our molecular analyses reveal that it is this species that became widespread in European inland waters in recent decades rather than T. trichiatus, which is apparently endemic to rivers and their estuaries along the north-eastern Black Sea, at the foothills of the Caucasus. The native range of C. ischnus is much broader, including both the Black Sea and Caspian Sea basins. Despite its broad native range, it appears that it does not overlap with that of T. trichiatus.

Species delimitation

All the single-locus species delimitation methods based on mitochondrial markers indicated that the T. trichiatus–C. ischnus clade is distinct from C. tenellus major (Fig. 3). However, most methods did not distinguish T. trichiatus from C. ischnus, although the latter was sometimes split into several molecular operational taxonomic units (MOTUs) by the BIN, GMYC and PTP approaches (Fig. 3). The BIN method was the least conservative, splitting T. trichiatus and C. ischnus into four and seven MOTUs, respectively. In contrast, the BFD method clearly supported the evolutionary distinctiveness of T. trichiatus, because the null hypothesis of conspecificity with C.ischnus was rejected with high support in all marker combinations (BF > 10; Table 1). The model in which C. ischnus comprised three additional species, beside T. trichiatus (red stars in Fig. 3), was also strongly supported against the null model of conspecificity. However, in comparison to the two-species model, it performed marginally worse with respect to nuclear markers (BF = 6.65), but marginally better with respect to the mitochondrial markers (BF = 8.79) or the mitochondrial and nuclear markers combined (BF = 6.59; Table 1). Therefore, based on the present evidence, we cannot conclude that C. ischnus comprises additional cryptic species.

Morphology

The PERMANOVA test revealed that all three focal taxa are morphologically distinct, irrespective of sex (Table 2). Morphospace analyses with PCA and clustering revealed that C. tenellus major and C. ischnus are more similar to one another than they are to T. trichiatus (Fig. 5A, B). The pattern was the same for both males and females. The first three PCA axes explained 69% of the variance for females and 70% for males. In general, T. trichiatus has relatively longer antennae and pereopods than C. ischnus (Fig. 5C).

SYSTEMATICS

Based on the preceding results, we propose the following taxonomic changes (see Discussion for justifications): (1) reassignment of Trichogammarus trichiatus (Martynov, 1932) to the genus Chaetogammarus as Chaetogammarus trichiatus; (2) synonymization of Trichogammarus with Chaetogammarus; and (3) resurrection and assignment of Chaetogammarus tenellus majorCărăușu, 1943 to a new genus, Spirogammarus, as Spirogammarus major.

We briefly redescribe and illustrate C. trichiatus, because this species was poorly illustrated in the original description by Martynov, whereas S. major and C. ischnus were well illustrated in previous works (Sars, 1896; Cărăușu, 1943; Cărăușu et al., 1955). We refer to Echinogammarus Stebbing, 1899 as s.l., in the broad sense of Pinkster (1993). However, detailed comparisons with the various new/resurrected genera proposed for various Echinogammarus clades (Hou & Sket, 2016; Sket & Hou, 2018) are presented in Supporting Information Table S4.

Order Amphipoda Latreille, 1816

 

Family Gammaridae Latreille, 1802

 

Genus Spirogammarus Copilaș-Ciocianu, Palatov, Marin & Grabowski gen. nov.

ZooBank registration:

urn:lsid:zoobank.org:act:D52696B2-AEBA-40DB-A7CC-BF712AAD3780

Type and only species:

Chaetogammarus tenellus majorCărăușu, 1943.

Diagnosis:

Large Echinogammarus-like gammarid amphipods (≤ 16 mm). Body smooth and non-carinate. Head anterolateral lobe straight. Antenna 1 poorly setose, accessory flagellum well developed (up to nine articles). Antenna 2 flagellum and peduncle segments densely set with tufts of setae twice as long as the width of underlying segment (distally coiled in males), calceoli absent. Gnathopod 1 propodus subequal to gnathopod 2 propodus. Posterior margin of carpal and meral (sometimes ischial) articles of pereopod 3 set with dense and distally coiled setae as long as/longer than the width of underlying segment; pereopods 4–7 with sparse and short setae, dominated by spines. Uropod 3 exopod with dense setae as long as/longer than the width of underlying segment (coiled in males); endopod short and rudimentary; pleonites unarmed dorsally; epimeral plates with spines only (second straight; third with produced inferoposterior corner); urosomites 1–2 without elevations, armed with four dorsolateral clusters of two to four spines. Telson fully cleft, as long as broad, armed with distal and lateral spines, with lobes abruptly tapering distally.

Etymology:

From the Latin spira, meaning coil/spiral, with reference to the coiled setae found on the antennae, gnathopods, pereopods and uropod 3 in males. The gender is masculine.

Remarks:

Spirogammarus can be distinguished readily from any species of Chaetogammarus and Echinogammarus s.l. by the presence of dense, distally coiled setae on the carpus and merus of pereopod 3 that are as long as/longer than the width of the underlying segment in both sexes. To our knowledge, no Echinogammarus-like taxa have females with coiled setae on the carpus and the merus of pereopod 3 (Pinkster, 1993). Spirogammarus is similar to Echinogammarus tibaldii Pinkster & Stock, 1970 with respect to the highly developed setosity of antenna 2, pereopod 3 and weakly setose pereopods 5–7 (Pinkster, 1993). However, Spirogammarus is distinguished by the weak setation of antenna 1 (highly developed), higher number of articles in the accessory flagellum (nine vs. five), coiled setae on gnathopod propodi and uropod 3 (non-coiled), and absence of long setae along the distal margin of epimeres. For further comparisons, see Supporting Information, Table S4.

Spirogammarus major (Cărăușu, 1943 ) comb. & stat. nov.

Chaetogammarus tenellus majorCărăușu, 1943.

Diagnosis:

Same as for genus (see above).

Remarks:

Spirogammarus major can be distinguished from C. trichiatus (in parentheses) by: its larger size (16 vs. 12 mm; Fig. 5C), absence of setae on the basal segment of mandibular palp, subequal gnathopod propodi (propodus 2 longer than propodus 1), straight anterolateral head lobes (acutely produced; Fig. 6B), denser setation along the inner side of the gnathopod palm (Fig. 6C), urosomites armed with clusters of two to four spines (solitary spines; Fig. 6A), uropod 3 exopod with dense setation in both sexes (only males have dense setation; Fig. 6D), and uropod 3 endopod in males is armed with spines and long setae (setae absent in both sexes; Fig. 6D). Both taxa are similar with respect to the setosity of antennae (Fig. 7A, B) and pereopod 3 (Fig. 7D).

Distribution:

Lagoons, estuaries and lower stretches of rivers that drain along the north-western to western shore of the Black Sea. Our morphomolecular analyses confirm that S. major is a widespread invasive species in European inland waters, being widely distributed along the Danube, lower Rhine, Oder and Dnieper rivers (Fig. 4).

Type locality:

Lake Shabla (Shablensko Ezero), Bulgaria.

Genus ChaetogammarusMartynov, 1924

Type species:

Gammarus ischnus Stebbing, 1899.

Included species:

Chaetogammarus hyrcanusPjatakova 1962, C. pauxillus (Sars, 1896), C. placidus (Sars, 1896), C. warpachowskyi (Sars, 1894) and C. trichiatusMartynov, 1932.

Non-included species:

Echinogammarus karadagiensisGrintsov, 2009 and E. mazestiensis Marin & Palatov, 2020 will be transferred to a new genus (I. Marin, D. Palatov, and D. Copilaș-Ciocianu, unpub. obs.).

Amended diagnosis:

Small- to large-sized species (≤ 15 mm); females smaller than males. Head with oblique anteroventral lobe, sometimes distally produced. Eyes large, reniform, well pigmented. Body non-carinate. Antenna 1 feebly setose, accessory flagellum two- to eight-segmented; antenna 2 usually shorter that antenna 1, without calceoli, deeply setose in males and less setose in females. Lower lip (labium) with mostly reduced inner lobes. Gnathopod 1 smaller than gnathopod 2, sexually dimorphic, stronger in males, propodus generally teardrop shaped; gnathopod 2 sexually dimorphic, stronger in males, propodus trapezoidal with oblique palmar margin. Basis of pereopods 5–7 without or with weak posteroventral lobes, dominated by spines. Uropod 3 with exopod about four to six times as long as wide, generally dominated by spines. Urosomites 1 and 2 without elevations, armed with clusters of spines; urosomite 3 with a pair of submedian spines. Telson deeply cleft into suboval lobes, bearing clusters of spines distally and submedially.

Synonymized genera:

TrichogammarusHou & Sket, 2016.

Distribution:

The native range of the genus is restricted to the Ponto-Caspian basin. Its species occur in the Caspian Sea at up to 500 m depth, and in lagoons, estuaries and lower stretches of rivers that drain into the Black, Azov and Caspian seas.

Chaetogammarus trichiatusMartynov, 1932

 

(Figs 8–11)

Brief redescription:

Based on specimen ZMMU Mb-1227, ♂, Russia, Krasnodar Krai, Sochi Urban Okrug, Lazarevsky District, mouth of the Ashe River, in the stream under stones (43°57.376ʹN, 39°15.954ʹE), 13 May 2019, coll. D. Palatov & I. Marin. Relatively large species (≤ 15 mm); females smaller than males. Head with oblique anteroventral lobe with distally produced anterior margin (Fig. 6B), eyes large, reniform, well pigmented. Body unpigmented, moderately elongated, generally smooth, non-carinate. Urosomites 1–3 smooth, without elevations; urosomites 1 and 2 with solitary strong median and submedian spines; urosomite 3 with two submedian spines and a pair of median simple setae (Fig. 6A). Antenna 1 feebly setose, with small aesthetascs; accessory flagellum eight-segmented (Figs 7A, 8A). Antenna 2 is about one-third shorter than antenna 1, massive and deeply setose in males and less setose in females, without calceoli and aesthetascs (Figs 7B, 8C, D). Upper lip (labrum) with convex distal part (Fig. 9A). Lower lip (labium) usually with mostly reduced inner lobes (Fig. 9B). Mandible with well-developed palp and setose basal segment (Fig. 9C–F). Maxilla 1 with outer lobe bearing hairbrush-like distal setae, subequal to inner lobe; inner lobe distally expanding with a row of marginal setae (Fig. 9G). Maxilla 2 with outer lobe wider than inner lobe, bluntly expanding distally (Fig. 9I). Maxilliped with outer and inner plates wide (Fig. 9J). Gnathopod 1 is smaller than gnathopod 2, with weak sexual dimorphism, stronger in males; propodus (palm) teardrop shaped, with oblique palmar margin in males and females (Fig. 8E, F). Gnathopod 2 is significantly larger in males, rectangularly elongated, with straight palmar margin in both males and females (Fig. 8H, I). Basis (article 2) of pereopods 5–7 with feebly marked ventral lobes (Fig. 10D, F, G); pereopod 3 with highly developed coiled setation along posterior margin (Figs 7D, 10A); basis of pereopod 7 elongated, ~2.5 times as long as wide, without ventral lobe (Figs 7C, 10G). Pleopods with two elongated hooks and one or two thick bristles in retinacules (Fig. 11F, G). Uropod 3 exopod broad, furnished with clusters of long, distally coiled setae, about four to six times as long as wide, with reduced distal article furnished with numerous long, simple distal setae (Figs 6D, 11K, J). Epimeral plates 1–3 with sharply produced inferoposterior corners (Fig. 11A–C). Telson deeply cleft into suboval lobes, abruptly tapering distally, bearing clusters of strong, stout setae distally and submedially (Figs 6A, 11D, E).

Remarks:

Males of C. trichiatus can be distinguished from males of other species of Chaetogammarus by the highly developed coiled setation on pereopod 3 posterior margin and uropod 3 exopod, acutely produced anterolateral head lobes, and telson lobes abruptly tapering distally. Chaetogammarus trichiatus is most similar to C. ischnus, but differs from that species in the following traits: significantly more elongate appendages (Fig. 5C); epimeres 2 and 3 with more produced posterodistal corners; presence of coiled setae along the outer margin of uropod 3 exopod (Fig. 6D); longer basis of pereopod 7 (length = 3 × width vs. length = 2 × width; Fig. 7C).

Distribution:

Chaetogammarus trichiatus is endemic to lakes, lower (slow current) river stretches and river mouths along the north-eastern Black Sea coast, from Abrau to the Shahe River, and a separate population is known from New Athos (Fig. 4). Found only on substrates with pebbles or large stones and boulders, apparently avoiding sandy and silty substrates.

Type locality:

Estuarine part of Khosta River, Sochi area, Russia. Presently absent at its original type locality owing to anthropogenic influence and the urban reconstruction of the lower part of the river.

DISCUSSION

Our integrative analyses revealed that C. trichiatus is not conspecific with its junior synonym C. tenellus major, both being distinct with respect to morphology, molecules and geographical ranges, and diverged at least since the Late Miocene. The former species is endemic to the north-eastern Black Sea coast, whereas the latter is native along the north-western Black Sea coast, from where it invaded European inland waters via the Danube and Dnieper rivers. Owing to their superficial morphological similarity, C. tenellus major has been misidentified as C. trichiatus for more than half a century, since its synonymization in 1969. Below, we discuss the implication of our findings and justify our taxonomic decisions.

Species status of S. major, C. trichiatus and C. ischnus

The integrative taxonomic approach taken in the present study allows us to clarify the species status of S. major. We found that this taxon is morphologically, genetically and geographically distinct from any other species and therefore merits specific status. However, the taxonomic situation of C. trichiatus is less straightforward. Although it is morphologically more distant from C. ischnus than is S. major (Fig. 5A, B), it is phylogenetically nested in C. ischnus, forming a distinct mitochondrial lineage (Fig. 3). At the nuclear level, all the four markers revealed that the two taxa share some haplotypes (Fig. 2). These shared haplotypes might not necessarily indicate hybridization/introgression, because they could represent incomplete lineage sorting owing to the slow evolution of nuclear markers (Berner & Salzburger, 2015; Mamos et al., 2021). The incomplete lineage sorting scenario is also strengthened by the fact that the two taxa have non-overlapping geographical ranges, with C. trichiatus being situated peripatrically or even allopatrically relative to C. ischnus (Fig. 4), meaning that opportunities for genetic exchange are limited.

Although the single-locus species delimitation methods failed to delimit C. trichiatus correctly, the multilocus Bayesian coalescent approach using Bayes factors strongly indicated that C. trichiatus is evolutionary distinct from C. ischnus, regardless of the marker combination (Table 1). Indeed, multilocus coalescent species delimitation methods have been shown to have a higher resolution than single-locus methods because they can detect recent speciation better (Camargo et al., 2012; Fontaneto et al., 2015). Our molecular dating analyses confirm this recent speciation, indicating that C. trichiatus split from C. ischnus ~0.41 Mya. This timing corresponds to the cold glacial period between marine isotope stages (MIS) 11 and 13 (Lisiecki & Raymo, 2005). Given that the north-eastern Black Sea coast is known as the Colchis glacial refugium (Tarkhnishvili et al., 2012) harbouring many endemic amphipod/crustacean species (Sidorov & Samokhin, 2016; Blaha et al., 2021; Marin et al., 2021; Palatov & Marin, 2021; Rendoš et al., 2021), we hypothesize that C. trichiatus speciated in this refugial area during the ice age that spanned MIS11–13.

Based on current evidence, we conclude that C. trichiatus is a young species that arose via peripatric speciation. Its profound morphological differentiation from C. ischnus in functionally important traits (much longer antennae and walking appendages; Fig. 5C) is intriguing given their recent divergence and is likely to reflect strong selective pressure and ecological differentiation (Schluter, 2000).

The recognition of C. trichiatus as a distinct species raises issues regarding the taxonomic status of C. ischnus. The former taxon is molecularly nested within the latter, rendering it paraphyletic. Chaetogammarus ischnus also harbours evolutionarily distinct mitochondrial lineages, some of which are even more divergent than C. trichiatus. However, the results of the Bayes factors species delimitation do not support the existence of cryptic species at the nuclear level, and support them only moderately at the mitochondrial level. Therefore, we consider that the evidence for cryptic species within C. ischnus is currently inconclusive, thus additional studies are necessary. We also caution that mitochondrial divergence alone should not be equated with species status in freshwater gammarids. This is because levels of mitochondrial divergence between species can sometimes be effectively zero (sympatric Gammarus in Lake Ohrid; Wysocka et al., 2013) and intraspecific variation can be as high as 20% with complete mixing of the nuclear genome (syntopic mitochondrial lineages of Echinogammarus sicilianus Karaman & Tibaldi, 1972; Hupało et al., 2022). Given this wide spectrum of mitochondrial distances, we consider that nuclear markers are more appropriate when defining cryptic gammarid species (Copilaș-Ciocianu et al., 2019b; Park & Poulin, 2022).

The Chaetogammarus problem

The genus Chaetogammarus initially comprised only Echinogammarus-like species endemic to the Ponto-Caspian basin (Martynov, 1924; i.e. C. ischnus, C. pauxillus, C. placidus and C. warpachowskyi). Later, Martynov (1932) added C. trichiatus and Pjatakova (1962) added C. hyrcanus. Subsequently, Stock (1968) expanded the genus to include other non-Ponto-Caspian species. Although Karaman synonymized Chaetogammarus with Echinogammarus, claiming morphological, ecological and geographical overlap (Karaman, 1977), this decision was not universally accepted and both names were used in the literature, causing further confusion (Stock, 1995). Copilaș-Ciocianu & Sidorov (2022) reinforced Martynov’s proposal by considering that only Ponto-Caspian Echinogammarus-like species should belong to Chaetogammarus. However, not all the Ponto-Caspian Chaetogammarus species were sequenced until the present study.

Our multilocus phylogenetic analyses encompassing all known species reveal that Chaetogammarus (as defined by Copilaș-Ciocianu & Sidorov, 2022) is polyphyletic, with its species being nested deep within the Ponto-Caspian gammaroid radiation, occupying various positions among morphologically disparate clades (Fig. 1). Moreover, Chaetogammarus species also vary greatly in morphology. As such, we deem it necessary to split this genus into several genera. Although a full reclassification of Chaetogammarus is beyond the scope of our study, we start by placing C. tenellus major in the newly erected monotypic genus Spirogammarus. This genus is highly distinct, both molecularly (Fig. 1) and morphologically (Supporting Information, Table S4), and its diagnosis is similar to Trichogammarus as defined by Hou & Sket (2016). However, given that the type species of Trichogammarus is C. trichiatus, and we find that it is nested within C. ischnus, Trichogammarus obviously becomes a junior synonym of Chaetogammarus. Moreover, although Trichogammarus was defined morphologically based on C. trichiatus, the DNA markers came from S. major (= C. tenellus major), which complicated the identity and validity of this genus further.

Among the other Chaetogammarus species that clearly need to be addressed are C. pauxillus and C. warpachowskyi, because they occupy distinct phylogenetic positions with respect to their congeners. The former seems to be an early-splitting lineage, but this could be an artefact owing to the fact that we successfully sequenced only two markers (EPRS and H3) from one individual. Additional material and further study are needed. In contrast, C. warpachowskyi has a well-defined phylogenetic position among species from the Gmelina group, with which it shares a number of morphological similarities. This species has always been taxonomically problematic and probably needs to be assigned to a new genus. Additionally, we find that the Pontic E. mazestiensis and E. karadagiensis are also nested in the Ponto-Caspian radiation and form a well-supported clade. We do not assign them to Chaetogammarus because they will be placed in a new genus in an upcoming study (I. Marin, D. Palatov, and D. Copilaș-Ciocianu, unpub. obs.). We, therefore, provisionally leave C. hyrcanus, C. pauxillus, C. placidus and C. warpachowskyi in Chaetogammarus pending further study.

Implications for monitoring and conservation

Our study highlights the importance of integrative taxonomy in the context of invasion biology. Given that taxonomy is the backbone of biology, proper taxonomic knowledge is fundamental for biodiversity monitoring and conservation (Giangrande, 2003; Vogel Ely et al., 2017). Furthermore, modern methods of biomonitoring (bulk metabarcoding or environmental DNA) rely on DNA barcoding, which, in turn, is dependent on the existence of reliable reference libraries (Weigand et al., 2019); therefore, it is important that taxa in these libraries are properly vetted taxonomically.

Here, we showed that the invasive species S. major has been misidentified as C. trichiatus in European inland waters for more than 50 years. Although S. major was previously included in molecular studies, none of the included populations was from the type locality or close to the type locality of C. trichiatus (Cristescu et al., 2004; Hou et al., 2014), hence this taxonomic error remained undetected. Our study has solved this taxonomic issue and provides validated DNA barcodes and morphological features to enable proper species identification.

Our study has redefined the areas of distribution of the focal species. Spirogammarus major is distributed natively throughout the north-western shore of the Black Sea and invaded European inland waters via the Danube and Dniester, reaching into the Rhine and Oder basins (Fig. 4). Chaetogammarus ischnus was originally described from the Caspian Sea from specimens collected at great depth, far from the shore. DNA analysis confirmed that C. ischnus is limited to the north-western coast of the Black Sea, in lagoons, estuaries and lower stretches of the Danube, Dniester and Dnieper, in addition to the Volga Delta (Fig. 4). There is one record of C. ischnus south of the distribution of C. trichiatus (question mark in Fig. 4), but these data are almost a century old and have never been confirmed by recent sampling (Copilaș-Ciocianu et al., 2020). Our detailed sampling at the mouth of the Volga River in 2021 did not encounter this species (I. Marin & D. Palatov, pers. comm.), although it was collected there two decades ago (Cristescu et al., 2004). All biotopes at the mouth of the Volga and north-western Caspian Sea are represented by silty/sandy bottoms, on which this species rarely occurs (Copilaș-Ciocianu & Sidorov, 2022). It is likely that in the Caspian Sea it should also be searched for on hard (rocky, boulder and pebble) bottoms. Also, we have not found any evidence (DNA data or museum collections) for the presence of C. ischnus in other locations on the territory of western Russia. For example, the amphipods identified as ‘Chaetogammarus ischnus/Gammarus ischnus’ and marked as Ponto-Caspian invaders were reported from the Gulf of Finland in the Baltic Sea (Berezina & Petryashev, 2012), from the Kuybyshev and Saratov reservoirs (Zinchenko & Kurina, 2011; Kurina, 2017) and the Lower Volga (Tarasova & Zaitsev, 2016). At present, we cannot confirm these identifications as С. ischnus, hence we view them as unconfirmed. Chaetogammarus trichiatus is currently found only in the basins of the small mountainous rivers flowing into the north-eastern Black Sea and its confirmed range does not overlap with that of C. ischnus (see Fig. 4).

Conclusion

Our study has clarified the taxonomic status of Trichogammarus and revealed that the widespread invader Spirogammarus major (formerly known as Chaetogammarus tenellus major) has been misidentified as Chaetogammarus/Echinogammarus trichiatus for more than half a century in European inland waters. Despite the two being superficially similar morphologically, they are phylogenetically distant, even belonging to different genera. Unexpectedly, we find that C. trichiatus is an incipient species molecularly nested in the widespread C. ischnus, despite profound morphological and geographical differentiation. The latter taxon also includes several distinct mitochondrial lineages that might represent cryptic species, warranting further research. In light of these results, we reassign T. trichiatus to Chaetogammarus, (C. trichiatus), synonymize Trichogammarus with Chaetogammarus and place the resurrected C. tenellus major in the new genus Spirogammarus. The multilocus molecular phylogeny also revealed that Chaetogammarus is polyphyletic, meaning that it needs to be split into additional genera. Our study highlights the importance of integrating multilocus DNA sequences, morphometry and biogeography in clarifying the status of taxonomically challenging groups, such as gammarid amphipods.

[Version of record, published online 15 April 2023; http://zoobank.org/urn:lsid:zoobank.org:pub:9B8367A2-727A-451E-A922-AD88CDEA5E66]

ACKNOWLEDGEMENTS

This project has received funding from the European Social Fund (project no. 09.3.3-LMT-K-712-19-0149) under a grant agreement with the Research Council of Lithuania (LMTLT). Additional material and data were obtained with the help of statutory funds of the Department of Invertebrate Zoology and Hydrobiology of University of Lodz, the National Science Centre (Poland) (contract no. 2017/01/X/NZ8/01086) and the European Commission’s Horizon 2020 research and innovation programme (contract no. 642973: Drivers of Pontocaspian Biodiversity Rise and Demise, PRIDE). T.R. was supported by a Scholarship of the Polish National Agency for Academic Exchange (NAWA) through the Bekker Programme (contract no. PPN/BEK/2018/1/00162). A.F.S. acknowledges support from the PRIDE programme.

We are most grateful to the Biodiversity and Systematic Collection, Justus Liebig University Giessen (Thomas Wilke and Christian Albrecht) and members or associates of the PRIDE team (Alberto Martínez Gándara, Ana Pavel, Elman Yusifov, Frank Wesselingh, Feodor Klimov, Konul Ahmadova, Manuel Sala Perez, Matteo Lattuada, Maxim Vinarski, Mikhail Son, Olga and Vitaliy Anistratenko, Sabrina van de Velde and Shebnem Feteliyeva Farzali), in addition to Agnieszka Rewicz, Eglė Šidagytė-Copilas, Halyna Morhun, Karolina Bącela-Spychalska, Péter Borza, Marek Michalski, Marius G. Berchi, Michael Zettler, Mykola Ovcharenko, Radomir Jaskuła, Sergiy Kudrenko, Teo Delić and Tomasz Mamos for their help with sampling and/or providing samples. We thank the two anonymous referees and the editors Maarten Christenhusz and Shane Ahyong for comments on the manuscript during the review process. We also thank Piotr Jóźwiak for his assistance with SEM photography and Dmitry Sidorov for providing important literature.

DATA AVAILABILITY

The newly obtained sequences are available in GenBank (COI: ON257949, ON257967, ON257994, ON258021 and OP466419–OP466517; 16S: ON258183, ON258198, ON258236 and OP466369–OP466418; 28S: OP466518–OP466576 and OP620914; H3: OP696981–OP697047; EPRS: OP466643–OP466708 and OP620915; and GAPDH: OP466577–OP466588). Molecular datasets generated and/or analysed during the present study are available in the BOLD repository (http://dx.doi.org/10.5883/DS-DCTRICH). Specimen data and raw morphometric measurements are available online in the Supporting Information files and Figshare (https://doi.org/10.6084/m9.figshare.21610182).

REFERENCES

SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article on the publisher's website:

Table S1. Information on samples used in the molecular analyses.

Table S2. Polymerase chain reaction primers and cycling protocols.

Table S3. Raw morphometric measurements (in millimetres).

Table S4. Diagnostic features separating the Echinogammarus-like genera