Shedding light on a cryptic cavernicole: A second species of Zenkevitchia Birstein (Crustacea, Amphipoda, Typhlogammaridae) discovered via molecular techniques

The Abkhazian region, in the southern foothills of the Caucasus Mountain Range, comprises a unique natural environment containing numerous subterranean habitats with relict and endemic lineages of obligate stygofauna. We aimed to assess the molecular phylogenetic relationships of Typhlogammaridae species from Balkan and Transcaucasian caves using the mitochondrial cytochrome c oxidase I (COI) in hopes of discovering previously undetected biodiversity. Our results showed molecular divergence within the genus Zenkevitchia Birstein, with two distinct groups located in the karstic regions Gudauta-Sukhumi and Gulripshi, respectively. These data indicated the existence of a new species (sequence divergences between groups of >14.3%) within the hitherto monotypic genus Zenkevitchia and allowed us to estimate the taxonomic relationship between Zenkevitchia admirabilis and Z. yakovi sp. n., based on examined morphological features and molecular phylogenetic relationships. We were unable to detect reliable morphological differences between Z. yakovi sp. n. and Z. admirabilis, highlighting the cryptic nature of the new species and the value of inclusion of molecular data in taxonomic studies.


Shedding light on a cryptic cavernicole:
A second species of Zenkevitchia Birstein (Crustacea, Amphipoda, Typhlogammaridae) discovered via molecular techniques

Introduction
Molecular identifications based on the mutability of DNA barcode region of the cytochrome c oxidase I (COI) of mitochondrion (Hebert et al. 2003) have advanced biodiversity inventories and resulted in the discovery of many new species (e.g., Smith et al. 2007), including amphipod taxa (Bradford et al. 2010).The Typhlogammaridae Bousfield, 1978 is the endemic Balkan-Transcaucasian family of cavernicole amphipods belonging to the huge Gammaridea suborder and represented in subterranean waters by 8 decribed species (and sub-species) in the region (Väinölä et al. 2008).The genus Zenkevitchia is monotypic, represented only by Zenkevitchia admirabilis Birstein, 1940 in Abkhazia.The species described as Zenkevitchia revazi Birstein & Ljovuschkin, 1970 has been transferred to the genus Anopogammarus (Karaman and Barnard 1979).Analyzing the geographical distribution of Zenkevitchia admirabilis is broadly distributed across the region, with published records across several karst areas: Gudauta region (Andreevskaya, Tarkiladze and Anakopiyskaya caves), Gulripshi district (Verkhne-, Sredne-, Nizhne-Shakuranskaya and Tsebeldinskaya caves) (Birstein 1940(Birstein , 1941;;Birstein and Ljovuschkin 1970), and the subterranean source of the Chernaya river near Gudauta (Derzhavin 1945).In his works, Birstein indicated that there was a wide range of morphological variability within Z. admirabilis.Carefully analysis of Birstein's works suggests that Z. admirabilis appears to be represented by a several morphotypes.In particular, the body size of adults varies widely.But characters which might indicate an excessive variability either were not specified by Birstein, or they were not clearly differentiated.The type series of Z. admirabilis has not been properly fixed according to the requirements of the Commission on Zoological Nomenclature in Code (1999: 75) (Article 72.2), as the type locality was not specified in the original description (Birstein 1940).In addition, the morphological and taxonomic boundaries of this species are vague, and thus the systematics of the genus Zenkevitchia appears to be in a state of confusion and in need of revision.Virtually nothing is known about the biology and origin of this enigmatic taxon.The heavily spinous outer plate of maxilla 1 implies a filtering function (Barnard and Barnard 1983).According to the data set and phylogenetic analysis of Hou et al. (2014) using nuclear 28S rDNA, the genus Zenkevitchia is sister to Anopogammarus Derzhavin, 1945, though this grouping has no bootstrap support.
In the present study, we sampled Zenkevitchia in limestone karstic caves located in the Shakuranian cave-complex near Tsabal in the Chjalta mountain range (southcentral Abkhazia).We evaluate the possible existence of hidden diversity within the genus Zenkevitchia using the DNA barcode region of the cytochrome c oxidase subunit I mitochondrial gene (COI) and evaluate the morphological traits of putative morphotypes.

Taxonomic sampling
Samples of blind, unpigmented amphipods were collected from two adjacent (4.1 km) localities in Abkhazia.All specimens were collected a fine-mesh hand-held dip net with a mesh size of 250 µm and fixed in 96% ethanol at the field site.

Morphology
A complete dissection was made of all pertinent morphological structures of specimens in the type series of the new species and permanent slide preparations were made using polyvinyl lactophenol (PVL) and methylene blue stain.Prior to dissection, body length was recorded by holding the specimen straight and measuring the distance along the dorsal side of the body from the base of the first antennae to the base of the telson.A Lomo MBS-9 stereomicroscope with a scaled micrometer eyepiece was used to make the measurements, and appendages were drawn using a Carl Zeiss NU-2 compound microscope equipped with a drawing device as modified by Gorodkov (1961).
To better understand the structure of outer plate of maxilla 1, and to facilitate counting the number of spines, the specimens were slightly crushed and squeezed in glycerol.The term "palmar angle" of the gnathopod propodi refers to the angle formed at the end of the palm and beginning of the posterior margin or the point at which the tip of the dactylus closes on the propodus (Birstein 1941); later the same structure was also named "defining angle" (Holsinger 1974), but we believe that the Birstein's combination of words better describes this feature.The nomenclature for setal patterns on article 3 of the mandibular palp follows Karaman (1970) and Stock (1974).The species diagnosis and taxonomic comparison were generated from a DELTA database (Dallwitz 2005) for the typhlogammarid genera and species of the world.
The description is based on the type series, with the material examined deposited in the Zoological Museum of the Far East Federal University, Vladivostok (FEFU; the holotype is here) and in the research collection of the Institute of Biology and Soil Science, Vladivostok (IBSS).

DNA extraction, PCR, sequencing and analyses
Total DNA was extracted from the muscle tissue with a DNeasy Blood & Tissue kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's guideline.The COI gene fragment was amplified using the universal primers HCO2198 and LCO1490 (Folmer et al. 1994).The annealing temperature was set to 40 °C for 20 s.The PCR products were sequenced directly using the same primers and a BigDye terminator v. 3.1 sequencing kit (Applied Biosystems, USA).Sequences were analyzed on an ABI 3130 genetic analyzer (Applied Biosystems, USA) and assembled with the Staden Package v. 1.4 (Bonfield et al. 1995).
The Akaike information criterion (AIC) in MODELGENERATOR 0.85 (Keane et al. 2006) was used to select the model of sequence evolution best fitting our data set (HKY+G).The data set was analyzed using the maximum-likelihood (ML) algorithm in SEAVIEW 4.0 (Gouy et al. 2010).A standard BioNJ initial tree was obtained automatically by applying the Nearest-Neighbor-Interchange (NNI) for tree inference.To assess support for clades 1,000 bootstrap replicates (Felsenstein 1985) were performed.

Data resources
The data underpinning the analysis reported in this paper are deposited in the Dryad Data Repository at http://dx.doi.org/10.5061/dryad.88fm6.

Molecular phylogenetic analysis
Taking into account reports of a rather wide range of body sizes of adults of Z. admirabilis from the region, we compared partial mitochondrial COI gene sequences for evaluation of the hidden diversity within the genus.The molecular analysis comprised 10 sequences of the family Typhlogammaridae of a total 428-620 bp in length available in GenBank (NCBI KF478522, KF478584, KF478590-KF478592, KF478599, KF478600), together with 3 individuals (NCBI KP844572-KP844574) representing of the newly described species from two adjacent (4.1 km) localities in Abkhazia.Phylogenetic relationships within Typhlogammaridae (Figure 1) were assessed using a data set consisting of all described species in the family besides Anopogammarus birsteini Derzhavin, 1945, a species inhabiting springs on the shores of Lake Goluboe (43.35087, 40.41115) near Gagry, Abkhazia, 80 km north of our study area.
Each amphipod specimen sequenced for this study along with referred in GenBank was characterized by a unique mitochondrial COI sequence excepts for both from the Sredne-Shakuranskaya Cave which are identical.Typhlogammarus mrazeki with other closer unidentified congener species accessions KF478590, KF478591 formed a strongly supported clade (95%) with Metohia carinata branching basally (97%).Relationship between unidentified Accubogammarus and Anopogammarus revazi remained unresolved in our analysis.Analysis of the mt-DNA barcode region proved to be an effective tool for the molecular identification and detection of cryptic diversity within Zenkevitchia inhabiting limestone caves in Abkhazia (Table 2), where two genetically distinct groups (Figure 1) were recovered.These groups are represented by specimens of Z. admirabilis collected in the Gudauta-Sukhumi region (Cluster B) and, in the Gulripshi region (Cluster A), by the new species describe below.The two groups showed genetic divergences ranging from 14.3% to 19.6% based on the uncorrected pairwise distances (Table 3).The monophyly of Zenkevitchia within the Typhlogammaridae is supported by moderate bootstrap values (62%) and the haplotypes for the two Zenkevitchia species differ from the rest of the Typhlogammaridae by the uncorrected pairwise distances mean sequence divergences of 45.4%.Zenkevitchia yakovi Sidorov, sp.n. http://zoobank.org/44C8C4D2-F34C-4F02-864A-08FE8D490B91Figures 2-7
Etymology.The specific epithet yakovi (Latin) refers to the first name of Yakov Avadievich Birstein a famous zoologist is known for his outstanding contributions to the systematics of subterranean crustaceans.

Distribution and ecology
Besides locus typicus of Z. yakovi sp.n. at the cave "Istočnik Tcebel'da" this species is known from Sredne-Shakuranskaya Cave, Gulripshi district of Abkhazia.The structure of the mouthparts, particularly unusual structure of outer plate with numerous long multi-toothed spines, suppose its filtering function.Analysis of the esophagus of a few individuals showed only the presence of detritus.
The nature of the morphological variability and divergence of COI gene sequences (pairwise distances 0.005) suggests that ,between the two caves "Istočnik Tcebel'da" and Sredne-Shakuranskaya, two populations of the same species are present.

Figure 1 .
Figure 1.Unrooted maximum-likelihood cladogram with bootstrap probabilities based on the mitochondrial cytochrome c oxidase I (COI) sequences for representatives of species within the family Typhlogammaridae.Specimen labels refer to information given in Table 2. Scale bar indicates the number of substitutions per site.

Figure 8 .
Figure 8. Habitat of Zenkevitchia yakovi sp.n. in the Sredne-Shakuranskaya cave, South Caucasus.One of the authors (DMP) sampling amphipods in the cave rivulet.

Table 1 .
Variation in morphological characters in adult specimens of Zenkevitchia yakovi sp.n. and comparison with published data for Z. admirabilis.

Table 2 .
List of the specimens, sampling sites and accession numbers of the sequences for COI included in this study.References are given for sequences obtained from GenBank.

Table 3 .
Estimates of pairwise sequence divergence (uncorrected p-distances) of partial mitochondrial COI gene among species and haplotypes of the Balkan and Caucasian Typhlogammaridae.